Compositions and methods for blood brain barrier delivery

Abstract

Monoclonal anti-TfR antibodies and antigen-binding fragments thereof for delivering agents to the brain of a subject in need thereof are described. Conjugates and fusion constructs containing anti-TfR antibodies or antigen-binding fragments thereof coupled to a therapeutic or diagnostic agent, e.g., a second antibody and antigen-binding fragments thereof, for treating or detecting neurological disorders and / or delivering therapeutic or diagnostic agents across the blood brain barrier are also described. Nucleic acids encoding the antibodies, conjugates, and fusion constructs and related recombinant host cells are also described.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 006,998, filed April 8, 2020, and U.S. Provisional Application No. 63 / 036,020, filed June 8, 2020, the disclosures of which are incorporated by reference herein in their entireties.

[0003] Reference to Electronically Submitted Sequence Listing

[0004] The instant application contains a Sequence Listing which has been submitted electronically via EFS-Web and is filed in ASCII format in compliance with the America Invention TECHNICAL FIELD

[0005] The present invention relates to blood brain barrier shuttling vectors that bind human transferrin receptor (TfR) and methods of use thereof. BACKGROUND

[0007] While the blood brain barrier (BBB) prevents harmful substances from entering the brain and is essential for brain homeostasis, it presents a formidable barrier to the effective delivery of drugs to the brain. Macromolecules, such as monoclonal antibodies and other biological therapeutics, have great therapeutic / diagnostic potential for treating / detecting pathologies in the central nervous system (CNS). However, their route into the brain is impeded by the BBB. Previous studies have shown that only a very small proportion (approximately 0.1%) of IgG injected into the bloodstream is able to cross the BBB into the CNS compartment (Felgenhauer, FEBS Lett. 52: 1158-1164, 1974)). This would limit any pharmacological effect due to the low concentration of antibodies within the CNS. Klin. Wschr

[0008] Many approaches have been investigated to improve brain delivery of therapeutic monoclonal antibodies (mAbs), including the use of receptor-mediated transcytosis (RMT). RMT utilizes receptors that are abundantly expressed on the luminal side of the BBB to transport across brain endothelial cells. Previous work to generate a clinically viable platform to deliver therapeutic mAbs into the brain has focused on antibody engineering to increase the efficiency of transcytosis, with benefits gained through observations in binding valency, pH dependence, and affinity (reviewed in Goulatis et al., 2017, Ther. Deliv. 8: 1-15). However, these approaches have not been able to achieve the desired levels of brain delivery for therapeutic mAbs. Curr Opin Struct Biol ​45: 109-115). However, translation to NHP and clinic was limited by rapid peripheral clearance from target-mediated drug disposition (TMDD) and safety from acute reticulocyte depletion (Gadkar, 2016, Eur J Pharm Biopharm. 2016 Apr;101:53-61). The transferrin receptor (TfR), particularly TfR1, mediates transport of iron-loaded transferrin (Tf) from blood to brain and iron-depleted Tf back to blood (Kawabata, Free Radical Biology & Medicine, 133, 46-54, 2019). Anti-TfR1 monoclonal antibodies have been used to deliver drugs to the brain (Burkhart et al Progress in neurobiology, 181, 101665, 2019). However, safety liabilities and poor pharmacokinetics (PK) of anti-TfR1 monoclonal antibodies have hampered their clinical development as BBB carriers.

[0009] Thus, there is a need for anti-TfR monoclonal antibodies or antigen-binding fragments thereof that can be used to shuttle drugs efficiently into the brain with improved safety and PK. SUMMARY

[0011] The present application relates to an optimized platform for brain delivery that takes into account not only the brain concentration of the delivered agent, e.g., a therapeutic monoclonal antibody (mAb), but also the therapeutic relevant profile of the mAb, including the mAb’s peripheral pharmacokinetics, safety, and pharmacodynamics. The platform utilizes a TfR binding molecule, particularly an antibody or antigen-binding fragment thereof that binds to the transferrin receptor (TfR), preferably human transferrin receptor 1 (huTfR1), wherein the TfR binding molecule has been optimized for transport function defined by the binding rate k a and dissociation rate k d values at both neutral pH of 6.8-7.8, e.g., physiological pH (e.g., 7.4), and acidic pH of 4.5-6.5, e.g., the acidic pH typically found in endosomal compartments.

[0012] The present inventors surprisingly found that the optimal values are not just the fastest binding rate k a and the slowest dissociation rate k dValues such as might be expected in a typical antibody-target interaction. That is, for this system, one does not necessarily want to use a molecule that "binds" and associates with TfR at a relatively high rate, and then dissociates from the TfR relatively slowly to have the longest-lived antibody-target complex. Rather, in one embodiment, the optimized trafficking function of the TfR binding agents described herein preferably has a k a rate at physiological pH (e.g., 7.4) and a faster dissociation rate k d rate at lower pH (e.g., pH 6.5 or 6.0) when compared to the k d .

[0013] In one general aspect, the present application describes an anti-TfR antibody or antigen-binding fragment thereof for use in delivering a therapeutic or diagnostic agent to the brain of a subject in need thereof, wherein the anti-TfR antibody or antigen-binding fragment thereof binds transferrin receptor (TfR), preferably human TfRl, with a dissociation constant K D D at neutral pH, preferably 1 nM to 500 nM, and a dissociation rate constant k -4 D of at least 10 -1 sec -4 , preferably 10 -1 sec -1 at acidic pH, preferably pH 5. d .

[0014] In some embodiments, the anti-TfR antibody or antigen-binding fragment thereof of claim 1 has a dissociation rate constant k -2 D of 2 x 10 -4 sec -1 , preferably 2.0 x 10 -3 sec -1 at neutral pH. d .

[0015] In another embodiment, the optimized trafficking function of certain TfR binding agents described herein preferably has a k 5 D of at least 1.05 x 10 a at physiological acidic pH (e.g., 7.4) and a k -3 D of at least 2.0 x 10 -1 s d or faster. a and k dThe parameters reflect only optimized transcytosis conditions and in no way limit our finding that TfR-mediated transport of certain molecules conjugated to certain TfR binders of the present text can occur outside of the preferred parameters.

[0016] In one general aspect, the present application relates to an antibody or antigen binding fragment thereof for use in delivering a pharmaceutical agent to the brain of a subject in need thereof, wherein the antibody or antigen binding fragment thereof binds to a transferrin receptor (TfR), preferably human transferrin receptor 1 (huTfRl), comprising

[0017] (1) a heavy chain variable region comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 and a light chain variable region comprising light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 have the following amino acid sequences:

[0018] (i) SEQ ID NOs: 292, 293, 294, 295, 296, and 297, respectively;

[0019] (ii) SEQ ID NOs: 279, 280, 281, 282, 283, and 284, respectively;

[0020] (iii) SEQ ID NOs: 29, 30, 31, 32, 33, and 34, respectively;

[0021] (iv) SEQ ID NOs: 57, 58, 59, 60, 61, and 62, respectively;

[0022] (v) SEQ ID NOs: 85, 86, 87, 88, 89, and 90, respectively;

[0023] (vi) SEQ ID NOs: 110, 111, 112, 113, 114, and 115, respectively;

[0024] (vii) SEQ ID NOs: 135, 136, 137, 138, 139, and 140, respectively;

[0025] (viii) SEQ ID NOs: 191, 192, 193, 194, 195, and 196, respectively;

[0026] (ix) SEQ ID NOs: 244, 245, 246, 247, 248, and 249, respectively;

[0027] (x) SEQ ID NOs: 263, 264, 265, 266, 267, and 268, respectively;

[0028] (xi) SEQ ID NOs: 345, 346, 347, 348, 349, and 350, respectively;

[0029] (xii) SEQ ID NOs: 355, 356, 357, 358, 359, and 360, respectively;

[0030] (xiii) SEQ ID NOs: 365, 366, 367, 368, 369, and 370, respectively;

[0031] (xiv) SEQ ID NOs: 375, 376, 377, 378, 379, and 380, respectively;

[0032] (xv) SEQ ID NOs: 385, 386, 387, 388, 389, and 390, respectively;

[0033] (xvi) SEQ ID NOs: 395, 396, 377, 398, 399, and 400, respectively;

[0034] (xvii) SEQ ID NOs: 405, 406, 407, 408, 409, and 410, respectively;

[0035] (xviii) SEQ ID NOs: 415, 416, 417, 418, 419, and 420, respectively;

[0036] (xix) SEQ ID NOs: 425, 426, 427, 428, 429, and 430, respectively;

[0037] (xx) SEQ ID NOs: 435, 436, 437, 438, 439, and 440, respectively;

[0038] (xxi) SEQ ID NOs: 445, 446, 447, 448, 449, and 450, respectively;

[0039] (xxii) SEQ ID NOs: 455, 456, 457, 458, 459, and 460, respectively;

[0040] (xxiii) SEQ ID NOs: 465, 466, 467, 468, 469, and 470, respectively;

[0041] (xxiv) SEQ ID NOs: 475, 476, 477, 478, 479, and 480, respectively;

[0042] (xxv) SEQ ID NOs: 485, 486, 487, 488, 489, and 490, respectively;

[0043] (xxvi) SEQ ID NOs: 495, 496, 497, 498, 499, and 500, respectively;

[0044] (xxvii) SEQ ID NOs: 505, 506, 507, 508, 509, and 510, respectively;

[0045] (xxviii) SEQ ID NOs: 515, 516, 517, 518, 519, and 520, respectively;

[0046] (xxix) SEQ ID NOs: 525, 526, 527, 528, 529, and 530, respectively;

[0047] (xxx) SEQ ID NOs: 535, 536, 537, 538, 539, and 540, respectively; or

[0048] (xxxi) SEQ ID NOs: 545, 546, 547, 548, 549, and 550, respectively; or

[0049] (2) a heavy chain single variable domain (VHH) comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 having the following amino acid sequences:

[0050] (i) SEQ ID NOs: 7, 8, and 9, respectively;

[0051] (ii) SEQ ID NOs: 317, 318, and 319, respectively;

[0052] (iii) SEQ ID NOs: 324, 325, and 326, respectively;

[0053] (iv) SEQ ID NOs: 331, 332, and 333, respectively; or

[0054] (v) SEQ ID NOs: 338, 339, and 340, respectively.

[0055] In certain embodiments, the present application relates to an anti-TfR VHH fragment comprising an amino acid sequence having at least 80%, e.g., at least 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 6, 316, 323, 330, or 337.

[0056] In other embodiments, the present application relates to an anti-TfR single chain variable fragment (scFv) comprising a heavy chain variable region covalently linked to a light chain variable region by a linker, preferably the linker has the amino acid sequence of SEQ ID NO: 314. More preferably, the scFv comprises an amino acid sequence having at least 80%, e.g., at least 85%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 278, 291, 28, 56, 84, 109, 134, 162, 190, 218, 243, 262, 344, 354, 364, 374, 384, 394, 404, 414, 424, 434, 444, 454, 464, 474, 484, 494, 504, 514, 524, 534, or 544.

[0057] Another aspect of the present application relates to a conjugate comprising an anti-TfR antibody or antigen binding fragment thereof of the present application coupled to a therapeutic or diagnostic agent, e.g., a drug for a neurological disorder or an agent for detecting a neurological disorder. Preferably, the therapeutic or diagnostic agent is a second antibody or antigen binding fragment thereof that binds a brain target.

[0058] In certain embodiments, the present application relates to a fusion construct comprising an anti-TfR antibody or antigen binding fragment thereof of the present application covalently linked to a second antibody or antigen binding fragment thereof that binds a brain target, e.g., a brain target selected from the group consisting of beta-secretase 1 (BACE1), beta amyloid (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase 6.

[0059] In certain embodiments, the fusion construct of the present application comprises a second antibody or antigen-binding fragment thereof that binds to Tau and comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 554-559, respectively. Preferably, the second antibody is a monoclonal antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 310 and a light chain having the amino acid sequence of SEQ ID NO: 311.

[0060] In one embodiment, the fusion construct of the present application comprises an anti-TfR antibody or antigen-binding fragment thereof of the present application, preferably an anti-huTfR1 VHH or scFv fragment, covalently linked via a linker to the carboxy terminus of only one of the two heavy chains of a second antibody or antigen-binding fragment thereof that binds to a brain target. Preferably, the linker has the amino acid sequence of SEQ ID NO: 312 or SEQ ID NO: 313.

[0061] In certain embodiments, each of the two heavy chains of the second antibody or antigen-binding fragment thereof comprises a modified constant heavy chain 3 (CH3) domain to promote formation of a heterodimer between the two heavy chains as compared to a wild-type CH3 domain. Any mutations that promote formation of a heterodimer between the two heavy chains can be used. Preferably, the modified CH3 domain of the first heavy chain comprises amino acid modifications at positions T350, L351, F405, and Y407, and the modified CH3 domain of the second heavy chain comprises amino acid modifications at positions T350, T366, K392, and T394. Preferably, the amino acid modification at position T350 is T350V, T350I, T350L, or T350M; the amino acid modification at position L351 is L351Y; the amino acid modification at position F405 is F405A, F405V, F405T, or F405S; the amino acid modification at position Y407 is Y407V, Y407A, or Y407I; the amino acid modification at position T366 is T366L, T366I, T366V, or T366M; the amino acid modification at position K392 is K392F, K392L, or K392M; and the amino acid modification at position T394 is T394W. More preferably, the modified heterodimeric CH3 domain of the first heavy chain comprises mutations T350V, L351Y, F405A, and Y407V, and the modified heterodimeric CH3 domain of the second heavy chain comprises mutations T350V, T366L, K392L, and T394W. Throughout the specification, the numbering of amino acid residues of an antibody is according to the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), unless otherwise explicitly stated.

[0062] In certain embodiments, the fragment crystallizable region (Fc region) of the second antibody or antigen-binding fragment thereof contains substitutions that alter (increase or decrease), preferably increase, binding of the second antibody or antigen-binding fragment thereof to the neonatal Fc receptor (FcRn). Preferably, the one or more mutations enhance binding at acidic pH, more preferably the Fc has the M252Y / S254T / T256E (YTE) mutations, wherein the numbering of amino acid residues is according to the EU index as shown in Kabat.

[0063] In certain embodiments, the fragment crystallizable region (Fc region) of the second antibody or antigen-binding fragment thereof contains substitutions that alter (increase or decrease), preferably increase, binding of the second antibody or antigen-binding fragment thereof to the neonatal Fc receptor (FcRn). Preferably, the one or more mutations enhance binding at acidic pH, more preferably the Fc has the M252Y / S254T / T256E (YTE) mutations, wherein the numbering of amino acid residues is according to the EU index as shown in Kabat.

[0064] In certain embodiments, the fusion construct of the present application comprises:

[0065] (1) a first heavy chain having an amino acid sequence that is at least 80%, e.g., at least 85%, 90%, 95%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 301, 304, 307, 285, 288, 298, 10, 13, 16, 19, 22, 25, 35, 38, 41, 44, 47, 50, 53, 63, 66, 69, 72, 75, 78, 81, 91, 94, 97, 100, 103, 106, 116, 119, 122, 125, 128, 131, 141, 144, 147, 150, 153, 156, 159, 169, 172, 175, 178, 181, 184, 187, 197, 200, 203, 206, 209, 212, 215, 225, 228, 231, 234, 237, 240, 250, 252, 256, 259, 269, 272, 275, 320, 327, 334, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, and 471;

[0066] (2) two light chains each independently having an amino acid sequence that is at least 80%, e.g., at least 85%, 90%, 95%, or 100% identical to an amino acid sequence selected from 302, 305, 308, 286, 289, 299, 11, 14, 17, 20, 23, 26, 36, 39, 42, 45, 48, 51, 54, 64, 67, 70, 73, 76, 79, 82, 92, 95, 98, 101, 104, 107, 117, 120, 123, 126, 129, 132, 142, 145, 148, 151, 154, 157, 160, 170, 173, 176, 179, 182, 185, 188, 198, 201, 204, 207, 210, 213, 216, 226, 229, 232, 235, 238, 241, 251, 253, 257, 260, 270, 273, 276, 321, 328, 335, 342, 352, 362, 372, 382, 392, 402, 412, 422, 432, 442, 452, 462, and 472, respectively; and

[0067] (3) a second heavy chain having an amino acid sequence that is at least 80%, e.g., at least 85%, 90%, 95%, or 100% identical to an amino acid sequence selected from 303, 306, 309, 287, 290, 300, 12, 15, 18, 21, 24, 27, 37, 40, 43, 46, 49, 52, 55, 65, 68, 71, 74, 77, 80, 83, 93, 96, 99, 102, 105, 108, 118, 121, 124, 127, 130, 133, 143, 146, 149, 152, 155, 158, 161, 171, 174, 177, 180, 183, 186, 189, 199, 202, 205, 208, 211, 214, 217, 227, 230, 233, 236, 239, 242, 252, 254, 258, 261, 271, 274, 277, 322, 329, 336, 343, 353, 363, 373, 383, 393, 403, 413, 423, 433, 443, 453, 463, and 473, respectively.

[0068] Another general aspect of the application relates to an isolated nucleic acid encoding an antibody or antigen binding fragment, conjugate, or fusion construct of the application. Also provided are vectors comprising an isolated nucleic acid of the application, host cells comprising the nucleic acid or the vector.

[0069] Another general aspect of the application relates to a method of producing an antibody or antigen-binding fragment, conjugate, or fusion construct of the application. The method comprises culturing a cell comprising a nucleic acid of the application under conditions wherein the antibody or antigen-binding fragment, conjugate, or fusion construct is produced, and recovering the antibody or antigen-binding fragment, conjugate, or fusion construct from the cell or cell culture.

[0070] Further provided is a pharmaceutical composition comprising a conjugate or fusion construct of the application and a pharmaceutically acceptable carrier.

[0071] Another general aspect of the application relates to a method of treating or detecting a neurological disorder in a subject in need thereof, comprising administering to the subject an effective amount of an anti-TfR antibody or antigen-binding fragment thereof, conjugate, or fusion construct of the application or a pharmaceutical composition thereof. Preferably, the neurological disorder is selected from the group consisting of neurodegenerative diseases (e.g., Lewy body disease, post-polio syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, spinocerebellar ataxia, spinal muscular atrophy), tauopathies (e.g., Alzheimer's disease and supranuclear palsy), prion diseases (e.g., bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob disease, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron diseases, and neuroaxonal dystrophic diseases (e.g., Canavan disease, Huntington's disease, neuronal ceroid-lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Hallervorden-Spatz syndrome, lafora disease, Rett syndrome, Wilson's disease, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia (e.g., Pick's disease and spinocerebellar ataxia), and cancer of the CNS and / or brain (e.g., brain metastasis from cancer elsewhere in the body).

[0072] Preferably, the antibody or antigen-binding fragment thereof, conjugate, fusion construct, or pharmaceutical composition of the application is administered intravenously.

[0073] Also described are methods of delivering a therapeutic or diagnostic agent to the brain of a subject in need thereof comprising administering to the subject a conjugate comprising a therapeutic or diagnostic agent coupled to an anti-TfR antibody or antigen-binding fragment thereof of the present application. Preferably, the therapeutic or diagnostic agent is a second antibody or antigen-binding fragment thereof that binds to a brain target. More preferably, administration of the therapeutic or diagnostic agent coupled to an anti-TfR antibody or antigen-binding fragment thereof of the present application to the brain of a subject results in reduced Fc-mediated effector functions and / or does not cause rapid reticulocyte depletion compared to administration of the therapeutic or diagnostic agent without coupling to an anti-TfR antibody or antigen-binding fragment thereof of the present application.

[0074] Yet another general aspect of the application relates to a method of inducing antibody-dependent phagocytosis (ADP) without stimulating the secretion of proinflammatory cytokines in a subject in need thereof comprising administering to the subject a complex comprising a therapeutic antibody or antigen-binding fragment thereof covalently conjugated, preferably, to an antigen-binding fragment thereof according to an embodiment of the application, wherein the therapeutic antibody or antigen-binding fragment thereof does not have effector function, e.g., the therapeutic antibody or antigen-binding fragment thereof comprises one or more amino acid modifications of positions L234, L235, D270, N297, E318, K320, K322, P331 and P329, e.g., one, two or three mutations of L234A, L235A and P331S, wherein the numbering of amino acid residues is according to the EU index as shown in Kabat. Preferably, the therapeutic antibody or antigen-binding fragment thereof specifically binds to tau aggregates.

[0075] Other aspects, features, and benefits of the present application will be apparent from the following disclosure, including the detailed description of the application and its preferred embodiments, as well as the appended claims. BRIEF DESCRIPTION OF DRAWINGS

[0077] The foregoing summary, as well as the following detailed description of the application, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the application, there is shown in the drawings a current preferred embodiment. It should be understood, however, that the application is not limited to the precise arrangements and instrumentalities shown in the drawings.

[0078] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0079] Figure 1 is a schematic representation of the tripoid mAb format (also referred to as TTP mAb) for the brain delivery platform.

[0080] Figure 2is an image showing internalization of the Tripod mAb in human brain endothelial cells. The Tripod mAb is stained red, the nucleus is blue, and actin is green.

[0081] Figure 3 is a graph showing pH-dependent binding, which is evaluated by comparing the off-rate at pH 7.4 with the off-rate at pH reduced to 6.5 and 6.0. If the off-rate is faster with reduced pH, the Tripod mAb scores positive.

[0082] Figure 4 is an image showing internalization of the Tripod mAb BBBB383 in human brain endothelial cells. The Tripod mAb is stained red, the nucleus is blue, and actin is green.

[0083] Figures 5A-5B is a graph showing plasma ( Figure 5A ) and brain ( Figure 5B ) PK of BBBB383 and BBBB426. Anti-BACE mAb containing brain shuttle vectors BBBB383 and BBBB426 are compared to BBBB456 (anti-BACE mAb without brain shuttle vector). Symbols represent the mean of 4 mice (4 and 24 hours) or 5 mice (72 hours).

[0084] Figure 6 is a graph showing Ab1-40 concentration in brain after treatment with BBBB383 and BBBB426. Anti-BACE mAb containing brain shuttle vectors BBBB383 and BBBB426 are compared to BBBB456 (anti-BACE mAb without brain shuttle vector). Symbols represent the mean of 4 mice (4 and 24 hours) or 5 mice (72 hours).

[0085] Figures 7A-7B is a graph showing plasma ( Figure 7A ) and brain ( Figure 7B ) PK of brain shuttle vector anti-BACE mAbs. Anti-BACE mAb containing brain shuttle vectors are compared to BBBB456 (anti-BACE mAb without brain shuttle vector, solid diamonds and dashed line). Each symbol represents the mean of two mice at each time point.

[0086] Figure 8 is a graph showing Ab1-40 concentration in brain after treatment with brain shuttle vector mAbs. Anti-BACE mAb containing brain shuttle vectors are compared to BBBB456 (anti-BACE mAb without brain shuttle vector, solid diamonds and dashed line). A dose-dependent decrease in AB levels is observed for all brain shuttle vectors except BBBB983. Each symbol represents the mean of two mice at each time point.

[0087] Figure 9 Figure 3 is a graph showing the internalization of tripedal mAb BBB-00489 in human brain endothelial cells. Tripedal mAb is stained red, and actin is green.

[0088] Figure 10 Figure 4 is a graph showing brain pharmacokinetics in cynomolgus monkeys. Cynomolgus monkeys were given 10 mg / kg of three TTP mAbs, BBBBl 134 and BBBBl 136 (left) and BBBBl 133 (right), intravenously, and compared to control mAb PT1B844. Brain exposures were measured 72 hours post-dose (n= 3 cynomolgus monkeys / mAb). Brain concentrations were determined across various regions and averaged across animals for mAbs. Each symbol represents a region of the brain.

[0089] Figure 11 Figure 5 is a graph showing brain concentrations of mAbs containing brain shuttle vectors compared to non-brain shuttle vector controls in different regions. Each point represents each animal (n=3).

[0090] Figure 12 Figure 6 is a graph showing i . v . Figure 7 is a graph showing plasma concentrations of administered mAbs at 4, 24, and 72 hours. All brain shuttle vector mAbs have faster clearance rates than non-brain shuttle vector mAbs. Each point represents each animal (n=3).

[0091] Figure 13 Figure 8 is a graph showing reticulocyte depletion observed for BBBBl 134, but not the other mAbs, during the cynomolgus monkey study, demonstrating the effect of Fc function on TfR binding mAbs and reticulocyte depletion.

[0092] Figures 14A-14C Figure 9: Brain pharmacokinetics and pharmacodynamics of tripedal mAbs in huTfR knock-in mice. A panel of tripedal mAbs (BBBBx) were given intravenously to human TfR knock-in mice at 10 mg / kg and brain exposures were evaluated at 24 hours compared to a control mAb:

[0093] Figure 14A Figure 10: A range of enhanced exposures were observed, from no enhancement (BBBB974, open squares) to 10.5x (BBBB978, open triangles) (n= 2 mice, symbols represent each individual animal, where bars represent the mean and error bars represent the standard deviation).

[0094] Figure 14B Figure 11: Tripedal mAb off-rate is well correlated with brain exposure, where neither too fast nor too slow off-rates were observed to be optimal.

[0095] Figure 14C : Evaluation of mAbs - brain pharmacodynamics of anti-BACE antagonist mAbs, and for all three-foot mAbs except BBBB983, strong PK / PD relationship was observed in brain. BBBB983 has enhanced brain exposure (5.5x) but similar concentration of Αβ 1-40 (Each triangle represents an individual). It is hypothesized that slow-neutral dissociation rate prevents diffusion to target in brain.

[0096] Figure 15 is a plot showing mAb-mediated uptake into microglial phagosomes. All brain shuttle mAbs promoted more efficient uptake into phagosomes compared to the non-brain shuttle mAb PT1B844. Within the brain shuttle mAbs, those with full effector function (BBBB1131, 1134, and 1046) were more efficient than those without effector function.

[0097] Figure 16 is a plot showing mAb-mediated uptake into macrophage phagosomes. All brain shuttle mAbs promoted more efficient uptake into phagosomes compared to the non-brain shuttle mAb B21M-IgGl.

[0098] Figures 17A-17F : Brain pharmacokinetics in cynomolgus monkeys confirm enhanced brain delivery of therapeutic mAbs.

[0099] Figure 17A : Intravenous administration of two three-foot mAbs, BBBB1134 and BBBB1136, and one control mAb, PT1B844, to cynomolgus monkeys at 10 mg / kg. Brain exposure was measured 72 hours post-dose (n = 3 cynomolgus monkeys / mAb, symbols represent each individual animal, where bars represent mean and error bars represent standard deviation). Enhanced brain exposure was observed in all brain regions evaluated for both brain shuttle mAbs.

[0100] Figure 17B : 7x and 11x brain concentration enhancement was observed for BBBB1134 and BBBB136, respectively, compared to control mAb.

[0101] Figure 17C : Plasma exposure over 72 hours confirms target-mediated drug disposition for three-foot mAbs, with accelerated clearance observed compared to control mAb. The three-foot mAbs differ in their binding affinity for FcRn, with BBBB1136 containing the high binding affinity "YTE" mutation; BBBB1136 (triangles) has approximately 2x enhanced plasma concentration at 72 hours compared to BBBB1134 (open squares).

[0102] Figure 17D: In vitro ADCC activity of triapine mAbs (BBBB1134 and BBBBB1136) compared to positive control BBBB175 (high affinity anti-TfR binding IgGl mAb) and negative control CNTO3930 (IgGl mAb that does not bind target cells). With both human and cynomolgus PBMC, BBBBB1134 - an IgGl mAb enabled robust ADCC of target cells. No ADCC activity was observed for BBBBB1136 - an IgGl mAb with silenced effector function.

[0103] Figure 17E : SPR binding data for BBBB1134 and BBBBB1136 to complement component 1q (C1q). BBBB1134 binds C1q while BBBBB1136 does not.

[0104] Figure 17F : Reticulocyte depletion was observed in cynomolgus monkey PK studies. No reticulocyte loss was observed 2 days post-dose for control mAbs or BBBBB1136, while robust depletion was observed following treatment with BBBBB1134 (symbols represent individual animals, bars represent mean and error bars represent standard deviation).

[0105] Figures 18A-18D : Brain and serum pharmacokinetics of repeated dosing of BBBBB1133 in cynomolgus monkeys and dose response:

[0106] Figure 18A : Cynomolgus monkeys were given BBBBB1133 intravenously at 2 mg / kg, 10 mg / kg or 30 mg / kg and brain exposure was evaluated 1, 7 or 15 days later (n=3 monkeys / mAb and time point. Symbols represent mean brain concentration and error bars represent standard deviation). Linear brain PK was observed between 2 and 10 mg / kg, but non-linear PK was observed between 10 and 30 mg / kg, suggesting that 30 mg / kg is a saturating dose for TfR.

[0107] Figure 18B : Serum concentrations of BBBBB1133 were measured throughout the study (1, 6 hours post-dose and days 1, 2, 4, 10 and 14). Linear pharmacokinetics was observed at all three doses. T 1 / 2 = 6 days.

[0108] Figure 18C: Cynomolgus monkeys were dosed intravenously with 2 mg / kg, 10 mg / kg or 30 mg / kg of BBBB1133 once weekly for three weeks. Brain exposures were evaluated at 1, 7, 15 or 21 days post-dose (n=3 monkeys / mAb and time point. Symbols represent mean brain concentrations and error bars represent standard deviations). Linear brain PK was observed between 2 and 10 mg / kg, but non-linear PK was observed between 10 and 30 mg / kg, suggesting that 30 mg / kg is a saturating dose for TfR. Cumulative evidence was observed at the 30 mg / kg dose.

[0109] Figure 18D : Serum concentrations of BBBB1133 were measured throughout the study (1, 6 hours post first dose and days 1, 2, 4, 10, 14, 14.02, 14.25, 15, 16, 18 and 21). Linear pharmacokinetics was observed at all three doses, with no evidence of PK tolerance upon repeat dosing.

[0110] Figures 19A-19C : Non-canonical, non-FcyR-mediated ADP promotes efficient phagocytosis of Tau aggregates by human microglia:

[0111] Figure 19A : To evaluate the potential of effector function impaired IgGl tripartite mAbs BBBB1133 and BBBB1136 to promote uptake of tau aggregates by microglia, human iPSC-derived microglia were incubated with mAbs and biotinylated phospho-tau oligomers labeled with streptavidin Alexa Flour 488 (AF488). Four hours post-incubation, cells were washed, fixed, permeabilized, stained and imaged using confocal microscopy. Cells containing tau aggregates co-localized with Lamp-1 stained lysosomes were quantified. BBBB1133 and BBBB1136 promoted more efficient uptake and lysosomal trafficking compared to anti-Tau WT IgGl mAb PT1B844.

[0112] Figure 19B : Uptake of tau oligomers was blocked with excess soluble TfR ECD but not by the addition of soluble Fc, confirming that uptake occurs through TfR.

[0113] Figure 19C: Human iPSc-derived microglia were incubated with Alexa Fluor 488-labeled phospho-Tau peptide (green) in the presence of PT1B844 or BBBBl 133 for 4 hours. After fixation, cells were stained with antibodies against clathrin, EEAl, Rabl7 or Lamp 1 and detected with Alexa Fluor 647-secondary antibodies (red). Cells were imaged using Perkin Elmer Opera Phenix 60x magnification confocal mode. Representative cell images of 2 pm levels are shown. Scale bar = 10 pm. Arrows point to areas of co-localization detailed in the insets. The third column for each phospho-Tau-antibody treatment is a merged result of the other two columns. Cells were also stained and imaged with DAPI to detect nuclei and with hcs Cellmask orange to detect cytoplasm (not shown).

[0114] Figures 20A-20E : Efficient phagocytosis of Tau aggregates derived from human AD patient brains by non-canonical non-FcyR-mediated ADP promotion:

[0115] Figure 20A : Human monocyte-derived macrophages were incubated with Tau aggregates and BBBBl 133 (open squares) and control anti-Tau mAb PT1B844 (circles). The amount of pTau remaining in the culture supernatant was quantified over time. Similar pTau degradation was observed until 8 hours, at which time PT1B844-mediated ADP stalled, while BBBBl 133-mediated ADP continued to promote degradation.

[0116] Figure 20B : Similar trends were observed using human iPSc-derived microglia, where BBBBl 133 (open squares) enabled more robust pTau degradation over time compared to PT1B844. The mechanism of BBBBl 133-mediated pTau degradation was confirmed to occur through TfR by blocking degradation using excess soluble TfRECD.

[0117] Figures 20C-20E : Cytokine concentrations were evaluated in supernatants from microglia experiments. PT1B844-mediated pTau ADP stimulation promoted release of pro-inflammatory cytokines TNF a ( Figure 20C ), IL6 ( Figure 20D ), and IL1 b ( Figure 20E ), while BBBBl 133 did not stimulate similar release.

[0118] Figure 21: Co-injection of PHF with indicated tau antibodies reduces induction of tau pathology:

[0119] Figure 21A Statistical significant difference in partial dependence of the model on Fc dependent activity confirmed by neutralization of mouse IgG2a by Tau.

[0120] Figure 21B Neutralization of Tau inoculum by both anti-Tau mAbs compared to isotype control. No statistical difference observed between the mAbs and TTP mAb with slightly improved neutralization by TTP mAb compared to the mAbs confirming that the non-canonical ADP mechanism is functional in vivo.

[0121] DETAILED DESCRIPTION

[0122] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is incorporated by reference herein in its entirety. Discussion of the references states herein is intended merely for purposes of providing a context for the present application. Nothing it is to be construed as an admission that any or all of the material it is part of the prior art.

[0123] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this application belongs. Otherwise, certain terms used herein have the meanings as set forth herein below. All patents, published patent applications and publications, referred to in this application are incorporated herein by reference in their entirety. It must be noted that as used herein and in the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0124] Unless otherwise indicated, any numerical values described herein, such as concentrations or concentration ranges, should be understood to be modified in all instances by the term "about." Accordingly, numerical values are generally included plus or minus 10% of the stated value. For example, a dose of 10 mg includes 9 mg to 11 mg. As used herein, the use of a number range expressly includes all possible subranges, all individual numerical values within the range, including integers and fractions of the values within such ranges, unless the context clearly indicates otherwise.

[0125] As used herein, the conjunctive term "and / or" between multiple recited elements is understood to include both the individual options and the combined options. For example, where two elements are conjoined by "and / or," a first option involves applicability of the first element without the second element. A second option involves applicability of the second element without the first element. A third option involves applicability of the first and second elements together. Any of these options is understood to fall within the meaning, and thus satisfy the requirement of the term "and / or" as used herein. Simultaneous applicability of more than one option is also understood to fall within the meaning, and thus satisfy the requirement of the term "and / or."

[0126] Throughout this specification and the following claims, unless the context otherwise requires, the word "comprise," and variations such as "comprises" and "comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" can be replaced by the term "containing" or "including" or sometimes by the term "having."

[0127] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude the presence of materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the preceding terms, whether used herein in the specification or in the claims, can be replaced by the term "consisting of' or "consisting essentially of' to alter the scope of the disclosure.

[0128] The term "antibody" herein is used in the broadest sense and specifically includes full length monoclonal antibodies, polyclonal antibodies, and, unless otherwise specified or made clear from the context, antigen binding fragments, antibody variants, and multispecific molecules thereof, so long as they display the desired biological activity. Generally, full length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen binding portions thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The general principles of antibody molecule structure and various techniques for producing antibodies are provided in, e.g., Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., (1988).

[0129] Full length antibodies can be assigned to different "classes" depending on the amino acid sequence of the constant domain of their heavy chains. There are five major classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0130] An "antibody" can also be a heavy chain single variable domain (VHH) antibody, also known as a heavy chain only antibody (HcAb), which lacks light chains and can be naturally produced by camelids or sharks. The antigen binding portion of a HcAb comprises a VHH fragment.

[0131] As used herein, the term "recombinant antibody" refers to an antibody expressed from a recombinant host cell comprising nucleic acid encoding the antibody (e.g., a chimeric, humanized, or human antibody, or antigen-binding fragment thereof). Examples of "host cells" used to produce recombinant antibodies include: (1) mammalian cells, e.g., Chinese hamster ovary (CHO), COS, myeloma cells (including YO and NSO cells), baby hamster kidney (BHK), HeLa, and Vero cells; (2) insect cells, e.g., sf9, sf21, and Tn5; (3) plant cells, e.g., plants belonging to the genus Nicotiana (e.g., Nicotiana benthamiana) and the like; (4) yeast cells, e.g., those belonging to the genus Saccharomyces (e.g., Saccharomyces cerevisiae) or Aspergillus (e.g., Aspergillus niger); (5) bacterial cells, e.g., Escherichia coli cells or Bacillus subtilis cells, and the like. Nicotiana tabacum Saccharomyces cerevisiae

[0132] ​​An "antigen-binding fragment" of an antibody is a molecule that comprises a portion of a full length antibody that is capable of detectably binding to an antigen, typically comprising one or more portions of at least a V H region. Antigen-binding fragments include multivalent molecules comprising one, two, three, or more antigen-binding portions of an antibody, as well as single chain constructs in which VL and V H regions, or selected portions thereof, are linked by synthetic linkers or by recombinant methods to form a functional antigen-binding molecule. An antigen-binding fragment can also be a single domain antibody (sdAb), also known as a nanobody, which is an antibody fragment consisting of a single monomeric variable antibody domain (VHH). While some antigen-binding fragments of antibodies can be obtained by actual fragmentation of larger antibody molecules (e.g., enzymatic cleavage), most are typically produced by recombinant techniques. Antibodies of the present invention can be prepared as full length antibodies or antigen-binding fragments thereof. Examples of antigen-binding fragments include Fab, Fab', F(ab)2, F(ab')2, F(ab)3, Fv (typically the VL and V H domains of a single arm of an antibody), single chain Fv (scFv, see, e.g., Bird et al., Science 1988; 242:423-426; and Huston et al. PNAS 1988; 85:5879-5883), dsFv, Fd (typically the V H and CHI domains), and dAb (typically the V H domain) fragments; V H, V L, V HH, and V-NAR domains; monovalent molecules comprising a single V H and a single V L chain; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al., Protein Eng 1997; 10:949-57); camel IgG; IgNAR; and one or more isolated CDRs or functional paratopes, where isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together to form a functional antibody fragment. Various types of antibody fragments have been described or reviewed in, e.g., Holliger and Hudson, Nat Biotechnol 2005; 23:1126-1136; WO2005040219 and published U.S. patent applications 20050238646 and 20020161201. Antibody fragments can be obtained using conventional recombinant or protein engineering techniques, and fragments can be screened for antigen-binding or other functions in the same manner as whole antibodies.

[0133] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of full-length antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods, 24: 107-117 (1992); and Brennan et al., Science, 229: 81 (1985)). However, these fragments can now be produced directly via recombinant host cells. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated from recombinant host cell cultures. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv). See WO 1993 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. For example, antibody fragments can also be "linear antibodies," e.g., as described in U.S. Patent No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

[0134] The term "antibody derivative" as used herein refers to a molecule comprising a full-length antibody or antigen-binding fragment thereof, wherein one or more amino acids are chemically modified or replaced. Chemical modifications useful for antibody derivatives include, e.g., alkylation, PEGylation, acylation, ester formation or amide formation, etc., e.g., for linking the antibody to a second molecule. Exemplary modifications include PEGylation (e.g., cysteine-PEGylation), biotinylation, radiolabeling, and conjugation to a second agent (e.g., a cytotoxic agent).

[0135] Antibodies herein include "amino acid sequence variants" having altered antigen-binding or biological activity. Examples of such amino acid alterations include antibodies with enhanced antigen affinity (e.g., "affinity matured" antibodies), and antibodies with altered Fc regions (if present), e.g., with altered (increased or decreased) antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) (see, e.g., WO 00 / 42072, Presta, L. and WO 99 / 51642, Idusogie et al.), and / or increased or decreased serum half-life (see, e.g., WO 00 / 42072, Presta, L.).

[0136] A "multispecific molecule" comprises an antibody or antigen-binding fragment thereof that is associated or linked to at least one other functional molecule (e.g., another peptide or protein, such as another antibody or a receptor ligand) to form a molecule that binds at least two different binding sites or target molecules. Exemplary multispecific molecules include bispecific antibodies and antibodies linked to soluble receptor fragments or ligands.

[0137] As used herein, the term "human antibody" is intended to include antibodies having variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences (i.e., are identical or essentially identical to such sequences). Furthermore, if the antibody contains a constant region, the constant region also "derives from" a human germline immunoglobulin sequence. The human antibodies of the application can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which the CDR sequences derive from another mammalian species, such as a mouse.

[0138] A "humanized" antibody is a human / non-human chimeric antibody that contains minimal sequence from non-human immunoglobulin. In most instances, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR residues are those of a human immunoglobulin sequence. The humanized antibody optionally also can comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992), WO 92 / 02190, U.S. Patent Application 20060073137, and U.S. Patent Nos. 6,750,325, 6,632,927, 6,639,055, 6,548,640, 6,407,213, 6,180,370, 6,054,297, 5,929,212, 5,895,205, 5,886,152, 5,877,293, 5,869,619, 5,821,337, 5,821,123, 5,770,196, 5,777,085, 5,766,886, 5,714,350, 5,693,762, 5,693,761, 5,530,101, 5,585,089, and 5,225,539.

[0139] The term "hypervariable region" when used herein refers to amino acid residues of an antibody which are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. U.S. Department of Health and Human Services, NIH Publication no. 91-3242) and / or those residues from a "hypervariable loop" (residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, J. Mol. Biol. 1987; 196:901-917). Generally, the amino acid residues are numbered by the method described in Kabat et al., supra. In this document, the phrase "Kabat position", "variable domain residue numbering by Kabat" and "according to Kabat" refer to this numbering system for the heavy chain variable domain or light chain variable domain. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insertion after residue 52 of CDR H2 (according to Kabat, residue 52a) and insertion residues (e.g. according to Kabat, residues 82a, 82b and 82c, etc.) after heavy chain FR residue 82. For a given antibody, the Kabat numbering of residues can be determined by alignment of the antibody sequence with the homologous regions of "standard" Kabat numbered sequences.

[0140] "Framework region" or "FR" residues are those variable domain residues other than the CDRs as defined herein.

[0141] "Epitope" or "binding site" is the region or site on an antigen to which an antigen-binding peptide (e.g. an antibody) specifically binds. A protein epitope can include amino acid residues that directly participate in binding (also known as the immunodominant component of the epitope) and other amino acid residues that do not directly participate in binding, e.g. amino acid residues that are effectively blocked by the specific antigen-binding peptide (in other words, amino acid residues that are within the "solvent excluded surface" and / or "footprint" of the specific antigen-binding peptide).

[0142] The "paratope" is the region or region of an antigen-binding portion of an antibody that specifically binds to an antigen. Unless otherwise indicated or contradicted by context, the paratope can include amino acid residues that directly participate in epitope binding, typically several of which are in the CDRs, as well as other amino acid residues that do not directly participate in binding, e.g., amino acid residues that are effectively blocked by the specifically bound antigen (in other words, amino acid residues that are within the "solvent-excluded surface" and / or "footprint" of the specifically bound antigen).

[0143] An "antibody that binds to the same epitope as a reference antibody" refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and vice versa.

[0144] An "isolated" antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for evaluating antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0145] With respect to the methods of the application, the term "administering" means a method of prophylactically or therapeutically preventing, treating or ameliorating a syndrome, disorder or disease described herein by using a conjugate or form thereof, composition or medicament of the application. Such a method includes administering an effective amount of the antibody, antigen-binding fragment thereof or conjugate or form thereof, composition or medicament simultaneously or at different times during the course of therapy or in combination. The methods of the application are understood to encompass all known therapeutic treatment regimens.

[0146] The ability of a target antibody to "block" the binding of a target molecule to a natural target ligand means that the antibody can detectably reduce the binding of the target molecule to the ligand in a dose-dependent manner in an assay using soluble or cell surface-associated target and ligand molecules, where the target molecule detectably binds the ligand in the absence of the antibody.

[0147] The "blood-brain barrier" or "BBB" refers to the physiological barrier between the peripheral circulation and the brain and spinal cord that is formed by tight junctions within the endothelial cell membranes of brain capillaries, creating a tight barrier that limits the transport of molecules into the brain. The BBB can limit transport of even very small molecules, such as urea (60 daltons), into the brain. Examples of the BBB include the BBB within the brain, the blood-spinal cord barrier within the spinal cord, and the blood-retinal barrier within the retina, all of which are continuous capillary barriers within the CNS. The BBB also includes the blood-CSF barrier (choroid plexus), where the barrier comprises ependymal cells, rather than capillary endothelial cells.

[0148] A "blood-brain barrier receptor" (referred to herein as "R / BBB") is an extracellular membrane-bound receptor protein expressed on brain endothelial cells that is capable of transporting molecules across the BBB or for use in transporting exogenously administered molecules. Examples of R / BBB include, but are not limited to, transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF-R), low-density lipoprotein receptor (including, but not limited to, low-density lipoprotein receptor-related protein 1 (LRP1) and low-density lipoprotein receptor-related protein 8 (LRP8)), and heparin-binding epidermal growth factor-like growth factor (HB-EGF). In the present context, an exemplary R / BBB is the transferrin receptor (TfR).

[0149] The "central nervous system" or "CNS" refers to the complex of nervous tissue that controls body functions and includes the brain and spinal cord.

[0150] As used herein, a "conjugate" refers to a protein covalently linked to one or more heterologous molecules, including, but not limited to, a therapeutic peptide or protein, an antibody, a tag, or a drug for a neurological disorder.

[0151] The term "coupled" as used herein refers to the joining or linking of two or more objects. When referring to chemical or biological compounds, coupled can refer to the covalent joining between two or more chemical or biological compounds. As a non-limiting example, an antibody of the present application can be coupled to a peptide of interest to form an antibody-conjugated peptide. An antibody-conjugated peptide can be formed by a specific chemical reaction designed to conjugate an antibody to a peptide. In certain embodiments, an antibody of the present application can be covalently coupled to a peptide of the present application by a linker. The linker can be, for example, covalently linked to the antibody or peptide first, and then covalently linked to the peptide or antibody.

[0152] An "effective amount" or "therapeutically effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0153] As used herein, "linker" refers to a chemical linker or a single chain peptide linker that covalently connects two different entities. The linker can be used to connect any two of the antibodies or fragments thereof, blood brain barrier shuttles, fusion proteins, and conjugates of the present application. The linker can connect, for example, the VH and VL in an scFv, or a monoclonal antibody or antigen binding fragment thereof to a therapeutic molecule, such as a second antibody. In some embodiments, if the monovalent binding entity comprises an scFv to TfR, preferably huTfRl, and the therapeutic molecule comprises an antibody to a CNS target, such as Tau, the linker can connect the scFv to the antibody to Tau. A single chain peptide linker comprising 1-25 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids linked by peptide bonds can be used. In certain embodiments, the amino acids are selected from the 20 naturally occurring amino acids. In certain other embodiments, one or more of the amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. Chemical linkers can also be used, such as hydrocarbon linkers, polyethylene glycol (PEG) linkers, polypropylene glycol (PPG) linkers, polysaccharide linkers, polyester linkers, hybrid linkers consisting of PEG and an embedded heterocyclic ring, and hydrocarbon chains.

[0154] As used herein, "neurological disorder" refers to a disease or condition affecting the CNS and / or having a CNS etiology. Exemplary CNS diseases or conditions include, but are not limited to, neuropathy, amyloidosis, cancer, an eye disease or condition, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, epilepsy, behavioral disorder, and lysosomal storage disorder. For purposes of this application, the CNS is understood to include the eye, which is generally isolated from the rest of the body by the blood-retinal barrier. Particular examples of neurological disorders include, but are not limited to, neurodegenerative diseases (including but not limited to Lewy body disease, post-polio syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, spinocerebellar ataxia, spinal muscular atrophy), tauopathies (including but not limited to Alzheimer's disease and supranuclear palsy), prion diseases (including but not limited to bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob disease, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, and neuroaxonal dystrophic diseases (including but not limited to Canavan disease, Huntington's disease, neuronal ceroid-lipofuscinosis, Alexander disease, Tourette syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Hallervorden-Spatz syndrome, Lafora disease, Rett syndrome, Wilson's disease, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia (including but not limited to Pick's disease and spinocerebellar ataxia), cancer (e.g., cancer of the CNS and / or brain, including brain metastasis from cancer elsewhere in the body).

[0155] A "neurological disorder drug" is a drug or therapeutic agent that can be used to treat or ameliorate the effects of one or more neurological disorders. Neurological disorder drugs of the present invention include, but are not limited to, small molecule compounds, antibodies, peptides, proteins, natural ligands of one or more CNS targets, modified forms of natural ligands of one or more CNS targets, aptamers, inhibitory nucleic acids (i.e., small inhibitory RNA (siRNA) and short hairpin RNA (shRNA)), ribozymes, or active fragments of any of the foregoing drugs. Exemplary neurological disorder drugs of the present invention are described herein and include, but are not limited to, antibodies, aptamers, proteins, peptides, inhibitory nucleic acids, and small molecules, and active fragments of any of the foregoing drugs, that are themselves or specifically recognize and / or act on (i.e., inhibit, activate, or detect) CNS antigens or target molecules, such as, but not limited to, amyloid precursor protein or portions thereof, beta amyloid, beta-secretase, gamma-secretase, tau, alpha-synuclein, parkin, huntingtin, DR6, presenilin, ApoE, glioma or other CNS cancer markers, and neurotrophic factors. Non-limiting examples of neurological disorder drugs and the corresponding disorders for which they can be used to treat: brain-derived neurotrophic factor (BDNF), chronic brain injury (neurogenesis), fibroblast growth factor 2 (FGF-2), anti-epidermal growth factor receptor, brain cancer, (EGFR)-antibody, glial cell line-derived neurotrophic factor, Parkinson's disease, (GDNF), brain-derived neurotrophic factor (BDNF), amyotrophic lateral sclerosis, depression, lysosomal enzymes, lysosomal storage disease of the brain, ciliary neurotrophic factor (CNTF), amyotrophic lateral sclerosis, neuregulin-1, schizophrenia, anti-HER2 antibody (e.g., trastuzumab), brain metastasis from HER2-positive cancer.

[0156] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to the subject to which the formulation would be administered.

[0157] As used herein, "pharmaceutically acceptable carrier or diluent" means any substance suitable for administration to an individual. For example, a pharmaceutically acceptable carrier can be a sterile aqueous solution, such as phosphate-buffered saline (PBS) or water for injection.

[0158] As used herein, "pharmaceutically acceptable salt" means a physiologically and pharmaceutically acceptable salt of a compound, e.g., an oligomeric compound or oligonucleotide, i.e., a salt that retains the desired biological activity of the parent compound and does not impart undesired pharmacological effects to the subject to which the salt is administered.

[0159] Pharmaceutically acceptable acidic / anionic salts used in the present application include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theochromate, tosylate, and trithiodide. Organic or inorganic acids also include, but are not limited to, hydriodic, perchloric, sulfuric, phosphoric, propionic, glycolic, methanesulfonic, hydroxyethanesulfonic, oxalic, 2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic, saccharinic, or trifluoroacetic acid. Pharmaceutically acceptable basic / cationic salts include, but are not limited to, aluminum, 2-amino-2-hydroxymethyl-propane-l,3-diol (also known as tris(hydroxymethyl)aminomethane, tromethamine, or “TRIS”), ammonia, benzathines, t-butyl amines, calcium, chloroprocaine, choline, cyclohexylamines, diethanolamines, ethylenediamines, lithium, L-lysine, magnesium, meglumine, N-methyl-D-glucamine, piperidine, potassium, procaine, quinine, sodium, triethanolamines, or zinc.

[0160] “Polypeptide” or “protein” means a molecule comprising at least two amino acid residues connected by a peptide bond to form a polypeptide. Small polypeptides of less than 50 amino acids can be referred to as “peptides.”

[0161] The phrases “sequence identity,” “percentage (%) sequence identity,” “% identity,” or “% identity with…” when used with respect to amino acid sequences describe the number of matches (“hit”) of the same amino acids in two or more aligned amino acid sequences compared to the number of amino acid residues that make up the full-length amino acid sequence. In other words, when comparing and aligning sequences for maximum consistency, as measured using sequence comparison algorithms known in the art, or when using manual alignment and visual inspection, the percentage of identical amino acid residues can be determined for two or more sequences (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identity for a full-length amino acid sequence). Sequences whose sequence identity is determined by comparison may therefore differ by substitutions, additions, or deletions of amino acids. Suitable procedures for aligning protein sequences are known to those skilled in the art. The percentage sequence identity of a protein sequence can be determined, for example, using programs such as CLUSTALW, Clustal Omega, FASTA, or BLAST, such as the NCBI BLAST algorithm (Altschul SF et al. (1997)). Nucleic Acids Res 25:3389-3402) confirmed.

[0162] The term "substantially identical" in the case of two amino acid sequences means that, when optimally aligned, such as by using the default gap weighting in programs like GAP or BESTFIT, the sequences share at least approximately 50% sequence identity. Typically, substantially identical sequences will show at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, at least approximately 95%, at least approximately 98%, or at least approximately 99% sequence identity.

[0163] "Specific binding," "specifically binding," or simply "binding" refers to an antibody binding to an antigen or an epitope within an antigen with an affinity greater than that for other antigens. Typically, antibodies bind at an affinity of approximately 1 x 10-1. -8 M or smaller, for example, about 1x10 -9 M or smaller, approximately 1x10 -10 M or smaller, approximately 1x10 -11 M or smaller or approximately 1x10 -12 M or a smaller dissociation constant (K) D ), usually due to its ability to bind non-specific antigens (e.g., BSA, casein) K D At most 1 / 100 of K D It binds to antigens or epitopes within antigens. K D k is the equilibrium dissociation constant between the antibody and its antigen. off / k on The ratio. K D It is negatively correlated with affinity. "Binding rate" (k...)on ) is a constant used to characterize how rapidly an antibody binds to its target. The "on-rate" (k off ) is a constant used to characterize how rapidly an antibody dissociates from its target. The dissociation constant K D can be measured using standard procedures. For example, the K D of an antibody can be determined by using surface plasmon resonance, for example by using a biosensor system, such as a Biacore® system, or by using a bio-layer interferometry technique, such as an Octet RED96 system. The K D The smaller the value, the higher the affinity of the antibody to bind to the target antigen. However, an antibody that specifically binds to an antigen or an epitope within an antigen can have cross-reactivity to other related antigens, such as the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (Macaca fascicularis, cyno), Pan troglodytes (Pan troglodytes, chimp), or Callithrix jacchus (Macaca mulatta, rhesus) monkey. While a monospecific antibody specifically binds to one antigen or one epitope, a bispecific antibody specifically binds to two different antigens or two different epitopes.

[0164] The term "subject" as used herein refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human. When the subject is a human, they can also be referred to as a "patient."

[0165] The term "transferrin receptor" or "TfR" as used herein refers to a cell surface receptor necessary for cellular iron uptake by receptor-mediated endocytosis. It is a carrier protein for transferrin. TfR is involved in iron uptake in vertebrates and is regulated in response to intracellular iron concentration. It imports iron by internalizing transferrin-iron complexes via receptor-mediated endocytosis. Two transferrin receptors have been characterized in humans, transferrin receptor 1 and transferrin receptor 2. Both of these receptors are transmembrane glycoproteins. TfR1 is a ubiquitously expressed high-affinity receptor. TfR2 binds transferrin with 1 / 25-1 / 30 the affinity of TfR1. Expression of TfR2 is restricted to certain cell types and is not influenced by intracellular iron concentration. In one embodiment, the TfR is a human TfR, which comprises an amino acid sequence as shown in, for example, Schneider et al. Nature 311 : 675-678 (1984). It can have a molecular weight of about 180,000 daltons, with two subunits each with an apparent molecular weight of about 90,000 daltons. Preferably, the TfR is human TfR1.

[0166] As used herein, a "target antigen" or "brain target" refers to an antigen and / or molecule expressed in the CNS (including the brain) that can be targeted by an antibody or small molecule. Examples of such antigens and / or molecules include, but are not limited to, beta-secretase 1 (BACE1), beta-amyloid (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase 6. In some embodiments, the target antigen is BACE1. In some embodiments, the target antigen is Tau.

[0167] As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") means clinical intervention in an attempt to alter the natural course of a treated individual and can be for prophylactic or during a clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the application are used to delay development of a disease, or to slow the progression of a disease.

[0168] Antibodies or immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the constant domain of the heavy chain. IgG is the most stable of the five types of immunoglobulins, with a serum half-life of about 23 days in humans. IgA and IgG are further subdivided into isotypes IgAl, IgA2, IgGl, IgG2, IgG3, and IgG4. The four IgG subclasses each have different biological functions, known as effector functions. These effector functions are generally mediated through interactions with Fc receptors (FcyR) and / or through binding of Clq and fixation of complement. Binding to FcyR can lead to antibody-dependent cell-mediated cytolysis or antibody-dependent cellular cytotoxicity (ADCC), while binding to complement factors can lead to complement-mediated cytolysis or complement-dependent cellular cytotoxicity (CDC). Anti-TfR antibodies of the application, or therapeutic or diagnostic antibodies conjugated or fused to anti-TfR antibodies, can have no or minimal effector functions, but retain their ability to bind FcRn, which binding can be the primary means by which the antibodies have an extended half-life in vivo.

[0169] Binding of FcγR or complement (e.g., Clq) to an antibody results from protein-protein interactions specified by so-called Fc moiety binding sites. Such Fc moiety binding sites are known in the art. Such Fc moiety binding sites include, for example, those characterized by amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to the EU index of Kabat). In some embodiments, an anti-TfR antibody of the application, or a therapeutic or diagnostic antibody conjugated or fused to an anti-TfR antibody, contains one or more substitutions at one or more Fc moiety binding sites to ablate effector function. For example, an anti-TfR antibody of the application, or a therapeutic or diagnostic antibody conjugated or fused to an anti-TfR antibody, can contain an Fc region that contains one or more of the following substitutions: a proline substitution for glutamic acid at residue 233, an alanine or valine substitution for phenylalanine at residue 234, and an alanine or glutamic acid substitution for leucine at residue 235 (EU numbering, Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. U.S. Dept. of Health and Human Services, Bethesda, Md., NIH Publication No. 91-3242). Preferably, an antibody of interest contains one, two, or three of the mutations L234A, L235A, and P331S (EU numbering, Kabat).

[0170] Antibodies of subclasses IgGl, IgG2, and IgG3 generally show complement activation, including Clq and C3 binding, whereas IgG4 does not activate the complement system and does not bind Clq and / or C3. In comparison to other IgG subclasses, the human IgG4 Fc region has a reduced ability to bind FcγR and complement factors. Preferably, an anti-TfR antibody of the application, or a therapeutic or diagnostic antibody conjugated or fused to an anti-TfR antibody, comprises an Fc region derived from a human IgG4 Fc region. More preferably, the Fc region contains a human IgG4 Fc region with substitutions that ablate effector function. For example, removal of the N-linked glycosylation site in the IgG4 Fc region by substitution of Ala for Asn at residue 297 (EU numbering) is another way to ensure ablation of residual effector function.

[0171] Anti-TfR antibodies and antigen-binding fragments thereof

[0172] In one general aspect, this application relates to antibodies or antigen-binding fragments thereof that bind to primate TfR, such as human TfR or monkey TfR, and said antibodies or antigen-binding fragments thereof are optimized to deliver a drug to the brain of a subject in need. The relationship between the binding affinity of anti-TfR antibodies to TfR and the efficiency of transcytosis has previously been described as transcytosis increasing with decreasing affinity for TfR (Yu, Zhang et al. 2011). Sci Transl Med 3(84):84ra44). The inventors of this invention have surprisingly discovered a more subtle relationship between affinity and transcytosis efficiency than previously described, where the effects of both binding rate and dissociation rate influence brain concentration. In particular, optimal brain PK and PD for agents (e.g., mAbs) that efficiently deliver anti-TfR antibodies or their antigen-binding fragments require a neutral dissociation rate that is neither too fast nor too slow.

[0173] Preferably, the anti-TfR antibody or its antigen-binding fragment of this application is pH-sensitive, for example, exhibiting different binding affinities to TfR at different pH values. For instance, the anti-TfR antibody of this application can bind to cell surface TfR with high affinity at neutral pH, such as physiological pH (e.g., pH 7.4), but dissociates from TfR at acidic pH, such as relatively low pH (pH 5.0-6.0), after internalization into the endosome compartment. Affinity is a measure of the strength of binding between two components, such as antibody and antigen. Affinity can be expressed in several ways. One way is based on the dissociation constant of the interaction (K). D K D It can be determined by conventional methods (including balanced dialysis) or by directly measuring the rate of antigen-antibody dissociation and association (kJ / kJ, respectively). off (kd or k) dis ) and k on (or ka) rate) measurement (see, for example, Nature, 1993 361:186-87). k off / k on The ratio eliminates all parameters unrelated to affinity and is equal to the dissociation constant K. D (See Davies et al. for general information) Annual Rev Biochem, 1990 59:439-473). Therefore, the smaller K D This implies a high degree of affinity. Another representation of affinity is K. a K D The reciprocal of, or k on / k off Therefore, a higher K aThis implies high affinity. For example, the antibody or its antigen-binding fragment used in the compositions and / or methods of this application may be used at a neutral pH (e.g., pH 6.8-7.8), such as physiological pH (e.g., pH 7.4), at a concentration of 1 nanomolar (nM, 10). −9 M) or larger K D Combined with TfR, and at acidic pH (e.g., pH 4.5–6.0), such as pH 5.0, at 10 -4 sec -1 or larger k dis Antibodies or fragments thereof dissociated from TfR.

[0174] Therefore, a general aspect of this application relates to anti-TfR antibodies or antigen-binding fragments thereof for delivering a drug to the brain of a subject in need, wherein the anti-TfR antibody or antigen-binding fragment thereof has a dissociation constant K of at least 1 nM, preferably from 1 nM to 500 nM, at neutral pH. D and at an acidic pH, preferably pH 5, for at least 10 -4 sec -1 Preferably 10 -4 Up to 10 -1 sec -1 dissociation rate constant k d It binds to the transferrin receptor (TfR), preferably human TfR1.

[0175] In one embodiment, the anti-TfR antibody or its antigen-binding fragment of this application has a 2 x 10⁻⁶ pH value at neutral pH. -2 Up to 2 x 10 -4 sec -1 For example, 2 x 10 -2 1 x 10 -2 9 x 10 -3 8 x 10 -3 7 x 10 -3 6 x 10 -3 5 x 10 -3 4 x 10 -3 3 x 10 -3 2 x 10 -3 1 x 10 -3 9 x 10 -4 8 x 10 -4 7 x 10 -4 6 x 10 -4 5 x 10 -4 4 x 10 -4 3 x 10 -4 2 x 10-4 sec -1 or any value in between d .

[0176] In certain embodiments, the antibody or antigen-binding fragment thereof that binds human TfR is a heavy chain single variable domain (VHH) antibody comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 having the following amino acid sequences:

[0177] (i) SEQ ID NOs: 7, 8 and 9, respectively;

[0178] (ii) SEQ ID NOs: 317, 318 and 319, respectively;

[0179] (iii) SEQ ID NOs: 324, 325 and 326, respectively;

[0180] (iv) SEQ ID NOs: 331, 332 and 333, respectively; or

[0181] (v) SEQ ID NOs: 338, 339 and 340, respectively.

[0182] Preferably, it is a VHH fragment comprising an amino acid sequence having at least 80%, e.g. at least 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 6, 316, 323, 330 or 337.

[0183] In other embodiments, the antibody or antigen-binding fragment thereof that binds human TfR comprises a heavy chain variable region comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the following amino acid sequences:

[0184] (i) SEQ ID NOs: 292, 293, 294, 295, 296 and 297, respectively;

[0185] (ii) SEQ ID NOs: 279, 280, 281, 282, 283 and 284, respectively;

[0186] (iii) SEQ ID NOs: 29, 30, 31, 32, 33 and 34, respectively;

[0187] (iv) SEQ ID NOs: 57, 58, 59, 60, 61, and 62, respectively;

[0188] (v) SEQ ID NOs: 85, 86, 87, 88, 89, and 90, respectively;

[0189] (vi) SEQ ID NOs: 110, 111, 112, 113, 114, and 115, respectively;

[0190] (vii) SEQ ID NOs: 135, 136, 137, 138, 139, and 140, respectively;

[0191] (viii) SEQ ID NOs: 191, 192, 193, 194, 195, and 196, respectively;

[0192] (ix) SEQ ID NOs: 244, 245, 246, 247, 248, and 249, respectively;

[0193] (x) SEQ ID NOs: 263, 264, 265, 266, 267, and 268, respectively;

[0194] (xi) SEQ ID NOs: 345, 346, 347, 348, 349, and 350, respectively;

[0195] (xii) SEQ ID NOs: 355, 356, 357, 358, 359, and 360, respectively;

[0196] (xiii) SEQ ID NOs: 365, 366, 367, 368, 369, and 370, respectively;

[0197] (xiv) SEQ ID NOs: 375, 376, 377, 378, 379, and 380, respectively;

[0198] (xv) SEQ ID NOs: 385, 386, 387, 388, 389, and 390, respectively;

[0199] (xvi) SEQ ID NOs: 395, 396, 377, 398, 399, and 400, respectively;

[0200] (xvii) SEQ ID NOs: 405, 406, 407, 408, 409, and 410, respectively;

[0201] (xviii) SEQ ID NOs: 415, 416, 417, 418, 419, and 420, respectively;

[0202] (xix) SEQ ID NOs: 425, 426, 427, 428, 429, and 430, respectively;

[0203] (xx) SEQ ID NOs: 435, 436, 437, 438, 439, and 440, respectively;

[0204] (xxi) SEQ ID NOs: 445, 446, 447, 448, 449, and 450, respectively;

[0205] (xxii) SEQ ID NOs: 455, 456, 457, 458, 459, and 460, respectively;

[0206] (xxiii) SEQ ID NOs: 465, 466, 467, 468, 469, and 470, respectively;

[0207] (xxiv) SEQ ID NOs: 475, 476, 477, 478, 479, and 480, respectively;

[0208] (xxv) SEQ ID NOs: 485, 486, 487, 488, 489, and 490, respectively;

[0209] (xxvi) SEQ ID NOs: 495, 496, 497, 498, 499, and 500, respectively;

[0210] (xxvii) SEQ ID NOs: 505, 506, 507, 508, 509, and 510, respectively;

[0211] (xxviii) SEQ ID NOs: 515, 516, 517, 518, 519, and 520, respectively;

[0212] (xxix) SEQ ID NOs: 525, 526, 527, 528, 529, and 530, respectively;

[0213] (xxx) SEQ ID NOs: 535, 536, 537, 538, 539, and 540, respectively; or

[0214] (xxxi) SEQ ID NOs: 545, 546, 547, 548, 549, and 550, respectively.

[0215] In other embodiments, the antibodies or antigen-binding fragments thereof of the present application compete with the antibodies or antigen-binding fragments exemplified herein. The binding site of an antibody or antigen can be determined by known methods, e.g., ELISA, Western blot, etc. In certain embodiments, such competing antibodies bind to the same epitope (e.g., linear or conformational epitope) bound by the exemplified antibodies or antigen-binding fragments thereof. Detailed exemplary methods for epitope mapping of antibody binding are provided in Morris, G. E., (ed.), “Epitope Mapping Protocols,” in: Methods in Molecular Biology, Vol. 66, Humana Press, Totowa, N.J. (1996). An “antibody that binds to the same epitope as a reference antibody” means an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of said antibody to its antigen in a competition assay by 50% or more.

[0216] Preferably, the antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv) comprising a heavy chain variable region (H V ) covalently linked to a light chain variable region (L V ) via a flexible linker. The scFv can retain the specificity of the original immunoglobulin, but with the constant regions removed and a linker introduced. In the scFv, the order of the domains can be H V - linker - L V , or L V - linker - H V . The linker can be newly designed, or derived from known protein structures to provide compatible length and conformation in bridging the variable domains of the scFv without serious steric hindrance. The linker can have a length of 10 to about 25 amino acids. Preferably, the linker is a peptide linker spanning about 3.5 nm (35 Å) between the carboxy terminus of the variable domain and the amino terminus of the other domain without affecting the ability of the domains to fold and form a complete antigen-binding site (Huston et al., Proc. Natl. Acad. Sci. USA, vol. 203, pp. 46-88, 1991, which is incorporated by reference herein in its entirety). The linker preferably comprises a hydrophilic sequence to avoid the peptide being embedded within or between the variable domains during the overall protein folding (Argos, EMBO J., vol. 7, pp. 1553-1559, 1988). Methods in Enzymology Journal of Molecular Biology ​, vol. 211, no. 4, pp. 943-958, 1990). For example, the linker can comprise Gly and Ser residues and / or together with interspersed charged residues such as Glu, Thr and Lys to enhance solubility. In one embodiment, the linker has the amino acid sequence of SEQ ID NO: 314 (GTEGKSSGSGSESKST). Any other suitable linker can also be used according to the present disclosure.

[0217] In some embodiments, the scFv comprises an amino acid sequence having at least 80%, e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 278, 291, 28, 56, 84, 109, 134, 162, 190, 218, 243, 262, 344, 354, 364, 374, 384, 394, 404, 414, 424, 434, 444, 454, 464, 474, 484, 494, 504, 514, 524, 534, or 544.

[0218] In preferred embodiments, the antibody or antigen binding fragment thereof binding to TfR, preferably human TfR1, does not contain a free cysteine.

[0219] According to the present disclosure, anti-TfR antibodies or antigen binding fragments thereof, e.g., VHH or scFv fragments, can be produced using suitable methods in the art. For example, VHH or scFv fragments can be recombinantly produced by culturing recombinant host cells (e.g., bacterial, yeast, or mammalian cells) under suitable conditions for production of the antibody fragments and recovering the fragments from the cell culture.

[0220] Brain Shuttle Vector Constructs

[0221] An optimized RMT brain delivery platform was developed using the transferrin receptor (TfR) by enhancing the efficiency of the intrinsic transcytosis, prolonging the peripheral pharmacokinetics and engineering for an acceptable safety profile while maintaining the efficacy of the therapeutic mAb. The interaction between transcytosis receptor affinity and brain concentration was investigated in human TfR knock-in mice. A comprehensive study of the binding kinetics confirmed that a neutral off-rate that is neither too fast nor too slow is required for optimal brain PK and PD of mAbs. The enhanced brain delivery observed in mice was confirmed in cynomolgus monkeys.

[0222] It was also found that engineered antibody constant regions with increased binding to the neonatal Fc receptor (FcRn) lead to reduced peripheral clearance and increased brain concentration.

[0223] Additional Fc mutations were introduced to abrogate binding to Fc gamma receptors (FcyR) and to avoid effector function-mediated toxicity. These mutations, when conjugated to high affinity anti-Tau binding mAbs, prevent effector function-mediated toxicity in the periphery while maintaining antibody-dependent phagocytosis (ADP) for microglial uptake and target degradation through a novel non-FcyR mechanism. This mechanism relies on internalization through TfR receptors and is more efficient than traditional FcyR-mediated ADP in promoting target degradation without stimulating pro-inflammatory cytokine secretion. To the best of the inventors' knowledge, this is the first report of non-FcyR-mediated ADP, representing a novel highly efficient non-inflammatory mechanism of phagocytosis that can be used for various therapeutic applications.

[0224] Thus, in one general aspect, the present application relates to an antibody-targeted brain delivery system comprising an anti-TfR antibody or antigen-binding fragment thereof of the present application. The anti-TfR antibody or antigen-binding fragment thereof can be used to deliver a therapeutic or diagnostic agent to a cell (e.g., a cancer cell) or BBB system. The agent that can be delivered includes any neurologic disorder drug or agent that can be used to detect or analyze a neurologic disorder drug. For example, such an agent can be a neurotrophic factor, including but not limited to nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), and insulin-like growth factor (IGF); a neuropeptide, including but not limited to substance P, neuropeptide Y, vasoactive intestinal peptide (VIP), gamma-aminobutyric acid (GABA), dopamine, cholecystokinin (CCK), endorphin, enkephalin, and thyrotropin-releasing hormone (TRH); a cytokine; an anxiolytic agent; an anticonvulsant; a polynucleotide and a transgene, including, for example, small interfering RNA and / or antisense oligomer; or an antibody or antigen-binding fragment thereof that binds to a brain target. The anti-TfR antibody or antigen-binding fragment thereof of the present application can be an effective tool to enhance delivery of an agent of interest from the blood to the brain and to function therein.

[0225] In particular, the agent of interest can be delivered parenterally, e.g., intravenously, in a combined form or linked to the anti-TfR antibody or antigen-binding fragment thereof of the present application. For example, the agent can be non-covalently linked to the anti-TfR antibody or antigen-binding fragment thereof. The agent can also be covalently linked to the anti-TfR antibody or antigen-binding fragment thereof to form a conjugate. In certain embodiments, conjugation is performed by constructing a protein fusion (i.e., by genetically fusing two genes encoding the anti-TfR antibody or antigen-binding fragment thereof and the neurologic disorder drug and expressing as a single protein). According to the present disclosure, the agent can be linked to the antibody or antigen-binding fragment thereof using known methods. See, e.g., Wu et al., 23(9): 1137-46, 2005; Trail et al., 17(3): 464-71, 1997; and US Patent No. 6,602,824. Nat Biotechnol , 23(9): 1137-46, 2005; Trail et al., 17(3): 464-71, 1997; and US Patent No. 6,602,824.Cancer Immunol Immunother ., 52(5):328-37, 2003; Saito et al, Adv Drug Deliv Rev ., 55(2):199-215, 2003; Jones et al, Pharmaceutical Research , 24(9):1759-1771, 2007.

[0226] In some embodiments, the therapeutic or diagnostic agent to be delivered to the brain and the anti-TfR antibody or antigen-binding fragment thereof can be covalently linked together (or conjugated) by a non-peptide linker or a peptide linker. Examples of non-peptide linkers include, but are not limited to, polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylenated polyols, polyvinyl alcohol, polysaccharides, dextrans, polyvinyl ethers, biodegradable polymers, polymeric lipids, chitin, and hyaluronic acid, or derivatives thereof or combinations thereof. The peptide linker can be a peptide chain consisting of 1-50 amino acids linked by peptide bonds, or derivatives thereof, whose N- and C-termini can be covalently linked to the anti-TfR antibody or antigen-binding fragment thereof.

[0227] In certain embodiments, the conjugates of the present application are multispecific antibodies comprising a first antigen-binding region that binds TfR and a second antigen-binding region that binds a brain antigen, e.g., beta-secretase 1 (BACE1), tau, and other brain antigens disclosed herein. Techniques for making multispecific antibodies include, but are not limited to, recombinant co- expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537, 1983), WO 93 / 08829 and Traunecker et al, EMBO J . 10: 3655, 1991) and “knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731,168). Multispecific antibodies can also be made by engineering electrostatic steering effects (WO 2009 / 089004 Al); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al, Science , 229: 81, 1985); using leucine zippers (see, e.g., Kostelny et al, J. Immunol ., 148(5): 1547-1553, 1992)); using the “diabody” technology (see, e.g., Hollinger et al, Proc. Natl. Acad. Sci. USA , 90:6444-6448, 1993)); using single-chain Fv (sFv) dimers (see, e.g., Gruber et al, J. Immunol, 152:5368 (1994)); and as described, e.g., in Tutt et al. J. Immunol . 147: 60, 1991 to prepare trispecific antibodies. The multispecific antibodies of the application also include antibodies having three or more functional antigen binding sites, including "Octopus antibodies" or "Dual Variable Domain Immunoglobulins" (DVD) (see, e.g., US 2006 / 0025576A1 and Wu et al. Nature Biotechnology , 25(11): 1290-7, 2007). The multispecific antibodies of the application also include "Dual Acting Fabs" or "DAFs," which comprise an antigen binding region that binds to TfR as well as a brain antigen (e.g., BACE1 or Tau) (see, e.g., US 2008 / 0069820). In one embodiment, the antibody is an antibody fragment, various such fragments are disclosed herein.

[0228] In one embodiment, the multispecific antibody of the application is a fusion construct comprising an anti-TfR antibody of the application or an antigen binding fragment thereof covalently linked (or fused) to a second antibody or antigen binding fragment thereof. Preferably, the second antibody or antigen binding fragment thereof binds to a brain target, e.g., BACE, tau or other brain antigen, such as those described herein. The anti-TfR antibody or antigen binding fragment thereof can be fused directly or via a linker to the carboxy and / or amino terminus of the light chain and / or heavy chain of the second antibody or antigen binding fragment thereof.

[0229] In one embodiment, the anti-TfR antibody or antigen binding fragment thereof is fused directly or via a linker to the carboxy terminus of the light chain of the second antibody or antigen binding fragment thereof.

[0230] In another embodiment, the anti-TfR antibody or antigen binding fragment thereof is fused directly or via a linker to the amino terminus of the light chain of the second antibody or antigen binding fragment thereof.

[0231] In another embodiment, the anti-TfR antibody or antigen binding fragment thereof is fused directly or via a linker to the carboxy terminus of the heavy chain of the second antibody or antigen binding fragment thereof.

[0232] In another embodiment, the anti-TfR antibody or antigen binding fragment thereof is fused directly or via a linker to the amino terminus of the heavy chain of the second antibody or antigen binding fragment thereof.

[0233] In preferred embodiments, the fusion construct of the present application comprises an anti-TfR antibody or antigen-binding fragment thereof of the present application, preferably an anti-huTfRl VHH or scFv fragment, covalently linked via a linker to the carboxy terminus of only one of the two heavy chains of a second antibody or antigen-binding fragment thereof that binds a brain target. Preferably, the linker has the amino acid sequence of SEQ ID NO: 312 or SEQ ID NO: 313.

[0234] To facilitate heterodimer formation between the two heavy chains, e.g., one with the fusion of an anti-TfR antibody or antigen-binding fragment thereof and one without, or one with Fc for the anti-TfR arm and one with Fc for the anti-brain target arm, heterodimer mutations are introduced into the Fc of both heavy chains. Examples of such Fc mutations include, but are not limited to, Zymeworks mutations (see, e.g., US 10,457,742) and “knob-in-hole” mutations (see, e.g., Ridgway et al, Protein Eng. , 9(7): 617-621, 1996). Other heterodimer mutations can also be used in the present application. In some embodiments, the modified CH3 described herein is used to facilitate heterodimer formation between the two heavy chains.

[0235] In addition to heterodimer mutations, other mutations can also be introduced. In some embodiments, the Fc region of the fusion construct or bispecific antibody further comprises one or more mutations that alter (increase or decrease), preferably eliminate, ADCC / CDC (e.g., the AAS mutations described herein) and / or one or more mutations that alter (increase or decrease), preferably increase, binding of the fusion construct or bispecific antibody to FcRn (e.g., the YTE mutations described herein). In some embodiments, one or more cysteine residues in the fusion construct or bispecific antibody are replaced with other amino acids, e.g., serine.

[0236] In certain embodiments, the fusion construct of the present application comprises:

[0237] (1) a first heavy chain having an amino acid sequence that is at least 80%, e.g., at least 85%, 90%, 95%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 301, 304, 307, 285, 288, 298, 10, 13, 16, 19, 22, 25, 35, 38, 41, 44, 47, 50, 53, 63, 66, 69, 72, 75, 78, 81, 91, 94, 97, 100, 103, 106, 116, 119, 122, 125, 128, 131, 141, 144, 147, 150, 153, 156, 159, 169, 172, 175, 178, 181, 184, 187, 197, 200, 203, 206, 209, 212, 215, 225, 228, 231, 234, 237, 240, 250, 252, 256, 259, 269, 272, 275, 320, 327, 334, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, and 471;

[0238] (2) two light chains each independently having an amino acid sequence that is at least 80%, e.g., at least 85%, 90%, 95%, or 100% identical to an amino acid sequence selected from the group consisting of 302, 305, 308, 286, 289, 299, 11, 14, 17, 20, 23, 26, 36, 39, 42, 45, 48, 51, 54, 64, 67, 70, 73, 76, 79, 82, 92, 95, 98, 101, 104, 107, 117, 120, 123, 126, 129, 132, 142, 145, 148, 151, 154, 157, 160, 170, 173, 176, 179, 182, 185, 188, 198, 201, 204, 207, 210, 213, 216, 226, 229, 232, 235, 238, 241, 251, 253, 257, 260, 270, 273, 276, 321, 328, 335, 342, 352, 362, 372, 382, 392, 402, 412, 422, 432, 442, 452, 462, and 472; and

[0239] (3) a second heavy chain having an amino acid sequence that is at least 80%, e.g., at least 85%, 90%, 95%, or 100% identical to an amino acid sequence selected from 303, 306, 309, 287, 290, 300, 12, 15, 18, 21, 24, 27, 37, 40, 43, 46, 49, 52, 55, 65, 68, 71, 74, 77, 80, 83, 93, 96, 99, 102, 105, 108, 118, 121, 124, 127, 130, 133, 143, 146, 149, 152, 155, 158, 161, 171, 174, 177, 180, 183, 186, 189, 199, 202, 205, 208, 211, 214, 217, 227, 230, 233, 236, 239, 242, 252, 254, 258, 261, 271, 274, 277, 322, 329, 336, 343, 353, 363, 373, 383, 393, 403, 413, 423, 433, 443, 453, 463, and 473, respectively.

[0240] According to the present disclosure, the conjugates, e.g., multispecific antibodies or fusion constructs, of the present application can be produced by any of a variety of techniques known in the art. For example, they can be expressed from recombinant host cells in which expression vectors encoding the heavy and light chains of the fusion constructs or multispecific antibodies have been transfected into the host cells by standard techniques. The host cells can be prokaryotic or eukaryotic host cells.

[0241] In an exemplary system, one or more recombinant expression vectors encoding the two heavy chains and light chains of the heterodimeric fusion constructs of the present application are introduced into host cells by transfection or electroporation. The selected transformant host cells are cultured under conditions sufficient to produce the fusion constructs to allow expression of the heavy and light chains, and the fusion constructs are recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vectors, to transfect the host cells, to select transformants, to culture the host cells, and to recover the protein constructs from the culture medium.

[0242] The present application provides isolated nucleic acids encoding the amino acid sequences of an anti-TfR antibody or antigen-binding fragment thereof, either alone or as part of a fusion construct or multispecific antibody as described in any of the embodiments or any of the claims herein. The isolated nucleic acids can be part of a vector, preferably an expression vector.

[0243] In another aspect, the application relates to host cells transformed with the vectors disclosed herein. In embodiments, the host cell is a prokaryotic cell, such as E. coli. In another embodiment, the host cell is a eukaryotic cell, such as a protist cell, an animal cell, a plant cell, or a fungal cell. In embodiments, the host cell is a mammalian cell, including but not limited to CHO, COS, NSO, SP2, PER.C6, or a fungal cell, such as S. cerevisiae, or an insect cell, such as Sf9.

[0244] Pharmaceutical compositions and related methods

[0245] The present application also relates to pharmaceutical compositions, methods of making and methods of using the same.

[0246] In another general aspect, the present application relates to a pharmaceutical composition comprising an anti-TfR antibody or antigen-binding fragment thereof or conjugate thereof of the present application and a pharmaceutically acceptable carrier. The anti-TfR antibody or antigen-binding fragment thereof or conjugate (e.g., a multispecific antibody or fusion construct) of the present application can also be used for the manufacture of a medicament for the therapeutic applications mentioned herein. The pharmaceutically acceptable carrier can be any suitable excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vehicle, microsphere, liposome encapsulation, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient, or diluent depend on the route of administration for a particular application.

[0247] Thus, in one embodiment, the present application relates to a method of transporting a therapeutic or diagnostic agent across the blood brain barrier (BBB) comprising exposing an anti-TfR antibody or antigen-binding fragment thereof coupled to the therapeutic or diagnostic agent to the BBB, such that the antibody or antigen-binding fragment thereof transports the agent coupled thereto across the BBB. In one embodiment, the agent is a neurological disorder drug. In another embodiment, the agent is an imaging agent or an agent for detecting a neurological disorder. Preferably, the anti-TfR antibody or antigen-binding fragment thereof or conjugate thereof does not impair the binding of TfR to its natural ligand, transferrin. The antibody specifically binds to TfR in a manner that does not inhibit the binding of TfR to transferrin. In some embodiments, the BBB is in a mammal, preferably a primate, such as a human, more preferably a human having a neurological disorder. In one embodiment, the neurological disorder is selected from the group consisting of Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman's syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, and traumatic brain injury.

[0248] In one embodiment, an anti-TfR antibody or antigen-binding fragment thereof or conjugate thereof of the present application is used to detect a neurological disorder prior to onset of symptoms and / or to assess the severity or duration of a disease or disorder. The antibody, antigen-binding fragment thereof or conjugate thereof allows detection and / or imaging of a neurological disorder, including imaging by radiography, tomography or magnetic resonance imaging (MRI).

[0249] In another embodiment, an anti-TfR antibody or antigen-binding fragment thereof or conjugate thereof is used to treat a neurological disorder (e.g., Alzheimer’s disease), which includes administering to a subject in need of treatment an effective amount of an anti-TfR antibody or antigen-binding fragment thereof or conjugate thereof. In some embodiments, the method further includes administering to the subject an effective amount of at least one additional therapeutic agent.

[0250] In another embodiment, the present application relates to the use of an anti-TfR antibody or antigen-binding fragment thereof or conjugate thereof of the present application in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treating a neurological disease or disorder. In a further embodiment, the medicament is for use in a method of treating a neurological disease or disorder, the method including administering to an individual having a neurological disease or disorder an effective amount of the medicament.

[0251] Another general aspect of the present application relates to a method of inducing antibody-dependent phagocytosis (ADP) without stimulating proinflammatory cytokine secretion in a subject in need thereof, which includes administering to the subject a complex comprising a therapeutic antibody or antigen-binding fragment thereof covalently conjugated, preferably covalently conjugated, to an antigen-binding fragment of an anti-TfR antibody according to embodiments of the present application, wherein the therapeutic antibody or antigen-binding fragment thereof does not have effector function. For example, the therapeutic antibody or antigen-binding fragment thereof can comprise one or more amino acid modifications that reduce or eliminate effector function, e.g., ADCC or CDC, such as mutations that reduce or abrogate binding to Fc gamma receptors. Such mutations can be one, two, or three mutations at positions L234, L235, D270, N297, E318, K320, K322, P331, and P329, e.g., L234A, L235A, and P331S, wherein the numbering of the amino acid residues is according to the EU index as shown in Kabat. In one embodiment, the therapeutic antibody or antigen-binding fragment thereof specifically binds to tau aggregates.

[0252] In some embodiments, the methods further comprise administering to the subject an effective amount of at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is one that is effective in treating the same or a different neurological disorder as the anti-TfR antibody or antigen-binding fragment or conjugate thereof being used to treat. Exemplary additional therapeutic agents include, but are not limited to, various neurological agents described above, cholinesterase inhibitors (e.g., donepezil, galantamine, rovastigmine, and tacrine), NMDA receptor antagonists (e.g., memantine), inhibitors of beta-amyloid peptide aggregation, antioxidants, gamma-secretase modulators, nerve growth factor (NGF) mimics or NGF gene therapy, PPARy agonists, HMS-CoA reductase inhibitors (statins), ampakines, calcium channel blockers, GABA receptor antagonists, glycogen synthase kinase inhibitors, intravenous immunoglobulin, muscarinic receptor agonists, nicotinic receptor modulators, active or passive beta-amyloid peptide immunization, phosphodiesterase inhibitors, serotonin receptor antagonists, and anti-beta-amyloid peptide antibodies. In certain embodiments, the at least one additional therapeutic agent is selected for its ability to reduce one or more side effects of a neurological agent. The additional therapeutic agent can be administered in the same or separate formulations, and together with or separately from the anti-TfR antibody or antigen-binding fragment or conjugate thereof. The anti-TfR antibody or antigen-binding fragment or conjugate thereof of the application can be administered prior to, concurrently with, and / or following administration of the additional therapeutic agent and / or adjuvant. The anti-TfR antibody or antigen-binding fragment or conjugate thereof of the application can also be used in combination with other interventional therapies, such as, but not limited to, radiation therapy, behavioral therapy, or other therapies known in the art and suitable for the neurological disorder to be treated or prevented.

[0253] The anti-TfR antibody or antigen-binding fragment or conjugate thereof of the application (as well as any additional therapeutic agents) can be administered by any suitable means, including parenterally, intrapulmonary, and intranasally, and, if desired for localized treatment, intralesionally. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration, depending partly on whether short- or long-term administration is involved. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at various time points, bolus administration, and pulsed infusion.

[0254] For the prevention or treatment of disease, the appropriate dosage of an anti-TfR antibody or antigen-binding fragment thereof or conjugate of the present application, when used alone or in combination with one or more other additional therapeutic agents, will depend on a variety of factors, e.g., the type of disease to be treated, the type of antibody or conjugate, the severity and course of the disease, whether the antibody, antigen-binding fragment thereof or conjugate is administered for preventive or therapeutic purposes, previous therapies, the patient's clinical history and response to the antibody, the subject's physical condition (e.g., age, weight, health), and the judgment of the treating physician. Optimal dosages will vary, but can be determined in a manner generally known to those skilled in the art. Antibodies, antigen-binding fragments thereof or conjugates are suitably administered to a patient at one time or over a series of treatments.

[0255] According to particular embodiments, a therapeutically effective amount refers to an amount of a therapy that is sufficient to achieve one, two, three, four or more of the following effects: (i) reducing or ameliorating the severity of the disease, disorder, or condition that is being treated or a symptom associated therewith; (ii) reducing the duration of the disease, disorder, or condition that is being treated or a symptom associated therewith; (iii) preventing progression of the disease, disorder, or condition that is being treated or a symptom associated therewith; (iv) causing regression of the disease, disorder, or condition that is being treated or a symptom associated therewith; (v) preventing development or onset of the disease, disorder, or condition that is being treated or a symptom associated therewith; (vi) preventing recurrence of the disease, disorder, or condition that is being treated or a symptom associated therewith; (vii) reducing hospitalization of a subject having the disease, disorder, or condition that is being treated or a symptom associated therewith; (viii) reducing length of hospitalization of a subject having the disease, disorder, or condition that is being treated or a symptom associated therewith; (ix) increasing survival of a subject having the disease, disorder, or condition that is being treated or a symptom associated therewith; (xi) inhibiting or reducing a disease, disorder, or condition that is being treated or a symptom associated therewith in a subject; and / or (xii) enhancing or improving the prophylactic or therapeutic effect of another therapy.

[0256] In another aspect, the application relates to the provision of articles of manufacture (e.g., kits) containing materials useful for treating, preventing and / or diagnosing the above conditions. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers can be formed from a variety of materials such as glass or plastic. The container holds a composition, which is by itself or in combination with another composition effective for treating, preventing and / or diagnosing the condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody, antigen-binding fragment thereof, or conjugate of the application. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture can comprise (a) a first container wherein is contained a composition comprising an antibody, antigen-binding fragment thereof, or conjugate of the application; and (b) a second container wherein is contained a composition comprising another cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the application can further include a package insert indicating that the compositions can be used to treat the particular condition. Optionally, the article of manufacture can further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, fillers, needles, and syringes.

[0257] Embodiments

[0258] The present application also provides the following non-limiting embodiments.

[0259] 1. An anti-TfR antibody or antigen-binding fragment thereof for use in delivering an agent to the brain of a subject in need thereof, wherein the anti-TfR antibody or antigen-binding fragment thereof binds to the transferrin receptor (TfR), preferably human TfRl, with a dissociation constant KD of at least 1 nM at neutral pH and a dissociation rate constant kd of at least 10 -4 sec -1 at acidic pH, preferably pH 5.

[0260] 1a. The anti-TfR antibody or antigen-binding fragment thereof of embodiment 1, having a dissociation constant KD of 1 nM to 500 nM, e.g., 1 nM, 10 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, or any value therebetween, at neutral pH.

[0261] 1b. The anti-TfR antibody or antigen-binding fragment thereof of embodiment 1 or 1a, having a dissociation rate constant kd of 10 -4 sec -1up to 10 -1 sec -1 for example 10 -4 , 10 -3 , 10 -2 , 10 -1 sec -1 or any value therebetween.

[0262] 2. The anti-TfR antibody or antigen-binding fragment thereof of any one of embodiments 1-1b, having an off-rate constant kd at neutral pH of 2 x 10 -2 to 2 x 10 -4 sec -1 , preferably 2.0 x 10 -3 sec -1 .

[0263] 2a. The anti-TfR antibody or antigen-binding fragment thereof of embodiment 2, wherein the off-rate constant kd at neutral pH is 2 x 10 -2 to 2 x 10 -4 sec -1 , for example 2 x 10 -2 , 1 x 10 -2 , 9 x 10 -3 , 8 x 10 -3 , 7 x 10 -3 , 6 x 10 -3 , 5 x 10 -3 , 4 x 10 -3 , 3 x 10 -3 , 2 x 10 -3 , 1 x 10 -3 , 9 x 10 -4 , 8 x 10 -4 , 7 x 10 -4 , 6 x 10 -4 , 5 x 10 -4 , 4 x 10 -4 , 3 x 10 -4 , 2 x 10 -4 sec -1 or any value therebetween.

[0264] 3. The anti-TfR antibody or antigen-binding fragment thereof of any one of embodiments 1-2a, comprising

[0265] (1) a heavy chain variable region comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 have the following amino acid sequences:

[0266] i. SEQ ID NOs: 292, 293, 294, 295, 296, and 297, respectively;

[0267] ii. SEQ ID NOs: 279, 280, 281, 282, 283, and 284, respectively;

[0268] iii. SEQ ID NOs: 29, 30, 31, 32, 33, and 34, respectively;

[0269] iv. SEQ ID NOs: 57, 58, 59, 60, 61, and 62, respectively;

[0270] v. SEQ ID NOs: 85, 86, 87, 88, 89, and 90, respectively;

[0271] vi. SEQ ID NOs: 110, 111, 112, 113, 114, and 115, respectively;

[0272] vii. SEQ ID NOs: 135, 136, 137, 138, 139, and 140, respectively;

[0273] viii. SEQ ID NOs: 191, 192, 193, 194, 195, and 196, respectively;

[0274] ix. SEQ ID NOs: 244, 245, 246, 247, 248, and 249, respectively;

[0275] x. SEQ ID NOs: 263, 264, 265, 266, 267, and 268, respectively;

[0276] xi. SEQ ID NOs: 345, 346, 347, 348, 349, and 350, respectively;

[0277] xii. SEQ ID NOs: 355, 356, 357, 358, 359, and 360, respectively;

[0278] xiii. SEQ ID NOs: 365, 366, 367, 368, 369, and 370, respectively;

[0279] xiv. SEQ ID NOs: 375, 376, 377, 378, 379 and 380, respectively;

[0280] xv. SEQ ID NOs: 385, 386, 387, 388, 389 and 390, respectively;

[0281] xvi. SEQ ID NOs: 395, 396, 377, 398, 399 and 400, respectively;

[0282] xvii. SEQ ID NOs: 405, 406, 407, 408, 409 and 410, respectively;

[0283] xviii. SEQ ID NOs: 415, 416, 417, 418, 419 and 420, respectively;

[0284] xix. SEQ ID NOs: 425, 426, 427, 428, 429 and 430, respectively;

[0285] xx. SEQ ID NOs: 435, 436, 437, 438, 439 and 440, respectively;

[0286] xxi. SEQ ID NOs: 445, 446, 447, 448, 449 and 450, respectively;

[0287] xxii. SEQ ID NOs: 455, 456, 457, 458, 459 and 460, respectively;

[0288] xxiii. SEQ ID NOs: 465, 466, 467, 468, 469 and 470, respectively;

[0289] xxiv. SEQ ID NOs: 475, 476, 477, 478, 479 and 480, respectively;

[0290] xxv. SEQ ID NOs: 485, 486, 487, 488, 489 and 490, respectively;

[0291] xxvi. SEQ ID NOs: 495, 496, 497, 498, 499 and 500, respectively;

[0292] xxvii. SEQ ID NOs: 505, 506, 507, 508, 509 and 510, respectively;

[0293] xxviii. SEQ ID NOs: 515, 516, 517, 518, 519, and 520, respectively;

[0294] xxix. SEQ ID NOs: 525, 526, 527, 528, 529, and 530, respectively;

[0295] xxx. SEQ ID NOs: 535, 536, 537, 538, 539, and 540, respectively; or

[0296] xxxi. SEQ ID NOs: 545, 546, 547, 548, 549, and 550, respectively; or

[0297] (2) a heavy chain single variable domain (VHH) comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 having the following amino acid sequences:

[0298] i. SEQ ID NOs: 7, 8, and 9, respectively;

[0299] ii. SEQ ID NOs: 317, 318, and 319, respectively;

[0300] iii. SEQ ID NOs: 324, 325, and 326, respectively;

[0301] iv. SEQ ID NOs: 331, 332, and 333, respectively; or

[0302] v. SEQ ID NOs: 338, 339, and 340, respectively.

[0303] 4. The antibody or antigen-binding fragment thereof of embodiment 3, which is a VHH fragment comprising an amino acid sequence having at least 80%, e.g., at least 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 6, 316, 323, 330, or 337.

[0304] 4a. The antibody or antigen-binding fragment thereof of embodiment 2, wherein the VHH fragment comprises the amino acid sequence of SEQ ID NO: 6, 316, 323, 330, or 337.

[0305] 5. The antibody or antigen-binding fragment thereof of embodiment 3, which is a single chain variable fragment (scFv) comprising a heavy chain variable region (VH) covalently linked to a light chain variable region (VL) by a linker, e.g., a peptide linker having a length of about 10 to about 25 amino acids.

[0306] 5a. The antibody or antigen-binding fragment thereof of embodiment 5, wherein in the scFv the VH is connected to the amino-terminal end of the VL by a linker.

[0307] 5b. The antibody or antigen-binding fragment thereof of embodiment 5, wherein in the scFv the VH is connected to the carboxy-terminal end of the VL by a linker.

[0308] 5c. The antibody or antigen-binding fragment thereof of embodiment 5a or 5b, wherein the linker comprises one or more of Gly and Ser, and one or more interspersed Glu, Thr and Lys residues, preferably the linker has the amino acid sequence of SEQ ID NO: 314.

[0309] 5d. The antibody or antigen-binding fragment thereof of embodiment 5c, wherein the scFv comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 279, 280, 281, 282, 283 and 284, respectively, or the amino acid sequences of SEQ ID NOs: 292, 293, 294, 295, 296 and 297, respectively.

[0310] 5e. The antibody or antigen-binding fragment thereof of embodiment 5, wherein the scFv comprises an amino acid sequence having at least 80%, e.g., at least 85%, 90%, 95% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 278, 291, 28, 56, 84, 109, 134, 162, 190, 218, 243, 262, 344, 354, 364, 374, 384, 394, 404, 414, 424, 434, 444, 454, 464, 474, 484, 494, 504, 514, 524, 534 or 544.

[0311] 5f. The antibody or antigen-binding fragment thereof of embodiment 5e, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 278, 291, 28, 56, 84, 109, 134, 162, 190, 218, 243, 262, 344, 354, 364, 374, 384, 394, 404, 414, 424, 434, 444, 454, 464, 474, 484, 494, 504, 514, 524, 534 or 544.

[0312] 5g. The antibody or antigen-binding fragment thereof of embodiment 5e, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 278, 291, 162 or 218.

[0313] 5h. An antibody or antigen-binding fragment thereof that binds to the same epitope as the antibody or antigen-binding fragment thereof of any one of embodiments 3-5g.

[0314] 5i. An antibody or antigen-binding fragment thereof that competes for binding to TfR with the antibody or antigen-binding fragment thereof of any one of embodiments 3-5g.

[0315] 5j. The antibody or antigen-binding fragment thereof of any one of embodiments 3-5i, which binds to human TfRl with a dissociation constant KD of 1 to 500 nM, e.g., 1 nM, 10 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, or any value in between, at pH 7.4.

[0316] 5k. The antibody or antigen-binding fragment thereof of any one of embodiments 3-5j, which binds to human TfRl with a dissociation rate constant kd of 10 -4 to 10 -1 sec -1 , e.g., 10 -4 , 10 -3 , 10 -2 , 10 -1 sec -1 or any value in between, at pH 5.

[0317] 6. A complex comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-5k coupled to a therapeutic or diagnostic agent.

[0318] 6a. The complex of embodiment 6, wherein the antibody or antigen-binding fragment thereof is non-covalently coupled to the therapeutic or diagnostic agent.

[0319] 6b. The complex of embodiment 6, wherein the antibody or antigen-binding fragment thereof is covalently coupled to the therapeutic or diagnostic agent to form a conjugate.

[0320] 6c. The complex of embodiment 6, wherein the antibody or antigen-binding fragment thereof is covalently linked to the therapeutic or diagnostic agent by a linker.

[0321] 6d. The complex of embodiment 6c, wherein the linker is a non-peptide linker, e.g., a polyethylene glycol, a polypropylene glycol, a copolymer of ethylene glycol and propylene glycol, a polyoxyethylated polyol, a polyvinyl alcohol, a polysaccharide, a dextran, a polyvinylether, a biodegradable polymer, a polymeric lipid, a chitin, and a hyaluronic acid or a derivative thereof, or a combination thereof.

[0322] 6e. The complex of embodiment 6c, wherein the linker is a peptide linker, e.g., a peptide chain consisting of 1-50 amino acids linked by peptide bonds or a derivative thereof.

[0323] 6f. The complex of any one of embodiments 6-6e, wherein the antibody or antigen binding fragment thereof is coupled to a diagnostic agent for detecting a neurological disorder, preferably the diagnostic agent is an agent for positron emission tomography (PET) or an agent for IDK.

[0324] 6g. The complex of any one of embodiments 6-6e, wherein the antibody or antigen binding fragment thereof is coupled to a therapeutic agent, preferably a neurological disorder drug.

[0325] 6h. The complex of embodiment 6g, wherein the neurological disorder drug is selected from the group consisting of small molecule compounds, antibodies, peptides, proteins, natural ligands of one or more CNS targets, modified forms of natural ligands of one or more CNS targets, aptamers, inhibitory nucleic acids (i.e., small inhibitory RNA (siRNA) and short hairpin RNA (shRNA)), ribozymes, and active fragments of the foregoing drugs.

[0326] 6i. The complex of embodiment 6g, wherein the neurological disorder drug is selected from the group consisting of antibodies, aptamers, proteins, peptides, inhibitory nucleic acids, and small molecules and active fragments of any of the foregoing drugs, which are themselves or specifically recognize and / or act on (i.e., inhibit, activate, or detect) CNS antigens or target molecules, such as but not limited to amyloid precursor protein or portions thereof, beta amyloid, beta-secretase, gamma-secretase, tau, alpha-synuclein, parkin, huntingtin, DR6, presenilin, ApoE, glioma or other CNS cancer markers, and neurotrophic factors. Non-limiting examples of neurological disorder drugs and the corresponding disorders they can be used to treat: brain-derived neurotrophic factor (BDNF), chronic brain injury (neurogenesis), fibroblast growth factor 2 (FGF-2), anti-epidermal growth factor receptor, brain cancer, (EGFR)-antibodies, glial cell line-derived neurotrophic factor, Parkinson’s disease, (GDNF), brain-derived neurotrophic factor (BDNF), amyotrophic lateral sclerosis, depression, lysosomal enzymes, lysosomal storage disorders of the brain, ciliary neurotrophic factor (CNTF), amyotrophic lateral sclerosis, neuregulin-1, schizophrenia, anti-HER2 antibodies (e.g., trastuzumab), brain metastasis from HER2-positive cancer.

[0327] 7. The complex of embodiment 6, which is a multispecific antibody comprising a first antigen binding region that binds TfR and a second antigen binding region that binds a brain antigen (or brain target), wherein the first antigen binding region comprises an antigen binding fragment thereof of any one of embodiments 1-5k.

[0328] 7a. The multispecific antibody of embodiment 7, wherein the brain target is selected from the group consisting of beta-secretase 1 (BACE1), beta amyloid (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase 6.

[0329] 7b. The multispecific antibody of embodiment 7a, wherein the second antigen binding region binds BACE1 or Tau.

[0330] 7c. The multispecific antibody of any one of embodiments 7-7b, wherein the first antigen binding region is covalently linked to a first Fc and the second antigen binding region is covalently linked to a second Fc.

[0331] 7d. The multispecific antibody of embodiment 7c, wherein the first Fc differs from the second Fc at one or more amino acid residues to promote formation of a heterodimer between the first Fc and the second Fc.

[0332] 8. The multispecific antibody of any one of embodiments 7-7d, which is a fusion construct of the antibody or antigen binding fragment thereof of any one of embodiments 1-5k comprising a second antibody or antigen binding fragment thereof covalently linked that binds a brain antigen (or brain target).

[0333] 8a. The fusion construct of embodiment 8, wherein the antibody or antigen binding fragment thereof of any one of embodiments 1-5k is covalently linked, preferably through a linker, to the amino-terminal end of the heavy chain of the second antibody or antigen binding fragment thereof.

[0334] 8b. The fusion construct of embodiment 8, wherein the antibody or antigen binding fragment thereof of any one of embodiments 1-5k is covalently linked, preferably through a linker, to the amino-terminal end of the light chain of the second antibody or antigen binding fragment thereof.

[0335] 8c. The fusion construct of embodiment 8, wherein the antibody or antigen binding fragment thereof of any one of embodiments 1-5k is covalently linked, preferably through a linker, to the carboxy-terminal end of the light chain of the second antibody or antigen binding fragment thereof.

[0336] 8d. The fusion construct of embodiment 8, wherein the antibody or antigen binding fragment thereof of any one of embodiments 1-5k is covalently linked, preferably through a linker, to the carboxy-terminal end of the heavy chain of the second antibody or antigen binding fragment thereof.

[0337] 9. The fusion construct of embodiment 8d, wherein the antibody or antigen-binding fragment thereof of any one of embodiments 1-5k is covalently linked to the carboxy-terminus of either of the two heavy chains of the second antibody or antigen-binding fragment via a linker.

[0338] 9a. The fusion construct of any one of embodiments 8a-9, wherein the linker is a peptide linker comprising one or more of Gly and Ser, preferably the linker has the amino acid sequence of SEQ ID NO: 312 or SEQ ID NO: 313.

[0339] 9b. The fusion construct of any one of embodiments 8-9a, wherein the second antibody or antigen-binding fragment thereof comprises a first Fc in its first heavy chain and a second Fc in its second heavy chain, and the first Fc differs from the second Fc at one or more amino acid residues to promote formation of a heterodimer between the first Fc and the second Fc.

[0340] 9c. The multispecific antibody of embodiment 7d or the fusion construct of embodiment 9b, wherein the first Fc contains one or more “knob” mutations and the second Fc contains one or more corresponding “hole” mutations, or vice versa (for “knob-into-hole” mutations, see, e.g., U.S. Patent No. 5,731,168; Ridgway et al., Protein Eng., 9(7): 617-621, 1996, which is incorporated by reference herein in its entirety), preferably a knob mutation of T366W and a hole mutation of T366S, L368A, or Y407V.

[0341] 9d. The multispecific antibody of embodiment 7d or the fusion construct of embodiment 9b, wherein the first Fc and the second Fc each comprise a modified heterodimeric CH3 domain compared to a wild-type CH3 domain polypeptide, preferably the modified heterodimeric CH3 domain comprises one or more mutations described in US 10,457,742.

[0342] 9e. The multispecific antibody or fusion construct of embodiment 9d, wherein the modified heterodimeric CH3 domain of the first Fc comprises amino acid modifications of positions T350, L351, F405, and Y407, and the modified heterodimeric CH3 domain of the second Fc comprises amino acid modifications of positions T350, T366, K392, and T394.

[0343] 9f. The multispecific antibody or fusion construct of embodiment 9e, wherein the amino acid modification at position T350 is T350V, T350I, T350L or T350M; the amino acid modification at position L351 is L351Y; the amino acid modification at position F405 is F405A, F405V, F405T or F405S; the amino acid modification at position Y407 is Y407V, Y407A or Y407I; the amino acid modification at position T366 is T366L, T366I, T366V or T366M; the amino acid modification at position K392 is K392F, K392L or K392M; and the amino acid modification at position T394 is T394W.

[0344] 9g. The multispecific antibody or fusion construct of embodiment 9e, wherein the modified heterodimeric CH3 domain of the first Fc comprises mutations T350V, L351Y, F405A and Y407V, and the modified heterodimeric CH3 domain of the second Fc comprises mutations T350V, T366L, K392L and T394W, or vice versa.

[0345] 10. The multispecific antibody or fusion construct of any one of embodiments 7-9g, wherein the Fc region of the multispecific antibody or fusion construct further comprises an alteration (increase or decrease), preferably an increase, in binding of the second antibody or antigen binding fragment thereof to the neonatal Fc receptor (FcRn).

[0346] 10a. The multispecific antibody or fusion construct of embodiment 10, wherein the second antibody or antigen binding fragment thereof comprises one or more mutations in the Fc domain that enhance binding of the fusion to the neonatal Fc receptor (FcRn).

[0347] 10b. The multispecific antibody or fusion construct of embodiment 10 or 10a, wherein the one or more mutations enhance binding at acidic pH.

[0348] 10c. The multispecific antibody or fusion construct of embodiment 10b, wherein the Fc of the second antibody has the M252Y / S254T / T256E (YTE) mutations, wherein the numbering of amino acid residues is in accordance with the EU index shown in Kabat.

[0349] 11. The multispecific antibody or fusion construct of any one of embodiments 7-10c, wherein the Fc region of the multispecific antibody or fusion construct further comprises an alteration (increase or decrease), preferably a decrease or ablation, in effector function.

[0350] 11a. The multispecific antibody or fusion construct of embodiment 11, wherein the second antibody or antigen binding fragment thereof comprises one or more mutations in the Fc domain that reduce or abrogate effector function, such as antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0351] 11b. The multispecific antibody or fusion construct of embodiment 11a, wherein the Fc of the second antibody has one or more amino acid modifications of positions L234, L235, D270, N297, E318, K320, K322, P331, and P329, wherein the numbering of amino acid residues is in accordance with the EU index as shown in Kabat.

[0352] 11c. The multispecific antibody or fusion construct of embodiment 11b, wherein the Fc of the second antibody has one, two, or three mutations of L234A, L235A, and P331S (AAS mutations).

[0353] 12. The multispecific antibody or fusion construct of any one of embodiments 7-11c, wherein the first antigen binding region or antibody or antigen binding fragment thereof does not contain a cysteine.

[0354] 13. The multispecific antibody or fusion construct of embodiments 7-12, wherein the second antigen binding region or second antibody or antigen binding fragment thereof binds to Tau, preferably comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 554-559, respectively, preferably the second antibody is a monoclonal antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 310 and a light chain having the amino acid sequence of SEQ ID NO: 311.

[0355] 14. The fusion construct of embodiment 9, comprising:

[0356] (1) a first heavy chain having an amino acid sequence that is at least 80%, e.g., at least 85%, 90%, 95%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 301, 304, 307, 285, 288, 298, 10, 13, 16, 19, 22, 25, 35, 38, 41, 44, 47, 50, 53, 63, 66, 69, 72, 75, 78, 81, 91, 94, 97, 100, 103, 106, 116, 119, 122, 125, 128, 131, 141, 144, 147, 150, 153, 156, 159, 169, 172, 175, 178, 181, 184, 187, 197, 200, 203, 206, 209, 212, 215, 225, 228, 231, 234, 237, 240, 250, 252, 256, 259, 269, 272, 275, 320, 327, 334, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, and 471;

[0357] (2) two light chains each independently having an amino acid sequence that is at least 80%, e.g., at least 85%, 90%, 95%, or 100% identical to an amino acid sequence selected from the group consisting of 302, 305, 308, 286, 289, 299, 11, 14, 17, 20, 23, 26, 36, 39, 42, 45, 48, 51, 54, 64, 67, 70, 73, 76, 79, 82, 92, 95, 98, 101, 104, 107, 117, 120, 123, 126, 129, 132, 142, 145, 148, 151, 154, 157, 160, 170, 173, 176, 179, 182, 185, 188, 198, 201, 204, 207, 210, 213, 216, 226, 229, 232, 235, 238, 241, 251, 253, 257, 260, 270, 273, 276, 321, 328, 335, 342, 352, 362, 372, 382, 392, 402, 412, 422, 432, 442, 452, 462, and 472; and

[0358] (3) a second heavy chain having an amino acid sequence that is at least 80%, e.g., at least 85%, 90%, 95%, or 100% identical to an amino acid sequence selected from 303, 306, 309, 287, 290, 300, 12, 15, 18, 21, 24, 27, 37, 40, 43, 46, 49, 52, 55, 65, 68, 71, 74, 77, 80, 83, 93, 96, 99, 102, 105, 108, 118, 121, 124, 127, 130, 133, 143, 146, 149, 152, 155, 158, 161, 171, 174, 177, 180, 183, 186, 189, 199, 202, 205, 208, 211, 214, 217, 227, 230, 233, 236, 239, 242, 252, 254, 258, 261, 271, 274, 277, 322, 329, 336, 343, 353, 363, 373, 383, 393, 403, 413, 423, 433, 443, 453, 463, and 473, respectively.

[0359] 14a. The fusion construct of embodiment 14, wherein the two light chains have the same amino acid sequence.

[0360] 14b. The fusion construct of embodiment 14, wherein the two light chains have different amino acid sequences.

[0361] 14c. The fusion construct of embodiment 14, wherein:

[0362] (1) the first heavy chain has an amino acid sequence selected from SEQ ID NO: 301, 304, 307, 285, 288, 298, 10, 13, 16, 19, 22, 25, 35, 38, 41, 44, 47, 50, 53, 63, 66, 69, 72, 75, 78, 81, 91, 94, 97, 100, 103, 106, 116, 119, 122, 125, 128, 131, 141, 144, 147, 150, 153, 156, 159, 169, 172, 175, 178, 181, 184, 187, 197, 200, 203, 206, 209, 212, 215, 225, 228, 231, 234, 237, 240, 250, 252, 256, 259, 269, 272, 275, 320, 327, 334, 341, 351, 361, 371, 381, 391, 401, 411, 421, 431, 441, 451, 461, and 471;

[0363] (2) the two light chains each have an amino acid sequence selected from 302, 305, 308, 286, 289, 299, 11, 14, 17, 20, 23, 26, 36, 39, 42, 45, 48, 51, 54, 64, 67, 70, 73, 76, 79, 82, 92, 95, 98, 101, 104, 107, 117, 120, 123, 126, 129, 132, 142, 145, 148, 151, 154, 157, 160, 170, 173, 176, 179, 182, 185, 188, 198, 201, 204, 207, 210, 213, 216, 226, 229, 232, 235, 238, 241, 251, 253, 257, 260, 270, 273, 276, 321, 328, 335, 342, 352, 362, 372, 382, 392, 402, 412, 422, 432, 442, 452, 462, and 472, respectively; and

[0364] (3) the second heavy chain has an amino acid sequence selected from 303, 306, 309, 287, 290, 300, 12, 15, 18, 21, 24, 27, 37, 40, 43, 46, 49, 52, 55, 65, 68, 71, 74, 77, 80, 83, 93, 96, 99, 102, 105, 108, 118, 121, 124, 127, 130, 133, 143, 146, 149, 152, 155, 158, 161, 171, 174, 177, 180, 183, 186, 189, 199, 202, 205, 208, 211, 214, 217, 227, 230, 233, 236, 239, 242, 252, 254, 258, 261, 271, 274, 277, 322, 329, 336, 343, 353, 363, 373, 383, 393, 403, 413, 423, 433, 443, 453, 463, and 473, respectively.

[0365] 14d. The fusion construct of embodiment 14, wherein:

[0366] (1) the first heavy chain has an amino acid sequence of SEQ ID NO: 285, 288, 298, or 301;

[0367] (2) the two light chains each have an amino acid sequence of 286, 289, 299, or 302, respectively; and

[0368] (3) the second heavy chain has an amino acid sequence of 287, 290, 300, or 303, respectively.

[0369] 15. An isolated nucleic acid encoding the antibody or antigen-binding fragment of any one of embodiments 1-5k or the fusion construct of any one of embodiments 7-14d.

[0370] 16. A vector comprising the isolated nucleic acid of claim 15.

[0371] 17. A host cell comprising the nucleic acid of embodiment 15 or the vector of embodiment 16.

[0372] 18. A method of producing the antibody or antigen-binding fragment of any one of embodiments 1-5k or the fusion construct of any one of embodiments 7-14d, comprising culturing a cell comprising a nucleic acid encoding the antibody or antigen-binding fragment or fusion construct under conditions wherein the antibody or antigen-binding fragment or fusion construct is produced, and recovering the antibody or antigen-binding fragment, conjugate, or fusion construct from the cell or cell culture.

[0373] 19. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of embodiments 1-5k, the complex of any one of embodiments 6-6i, or the multispecific antibody or fusion construct of any one of embodiments 7-14d, and a pharmaceutically acceptable carrier.

[0374] 20. A method of treating or detecting a neurological disorder in a subject in need thereof, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of any one of embodiments 1-5k, the complex of any one of embodiments 6-6i, or the multispecific antibody or fusion construct of any one of embodiments 7-14d, or the pharmaceutical composition of embodiment 19.

[0375] 21. A method of increasing delivery of a therapeutic or diagnostic agent to the brain of a subject in need thereof, comprising administering to the subject a conjugate comprising a therapeutic or diagnostic agent coupled to the antibody or antigen-binding fragment thereof of any one of embodiments 1-5k.

[0376] 22. A method of transporting a therapeutic or diagnostic agent across the blood-brain barrier (BBB), comprising exposing an anti-TfR antibody or antigen-binding fragment thereof of any one of embodiments 1-5k coupled to the therapeutic or diagnostic agent to the blood-brain barrier, such that the antibody or antigen-binding fragment thereof transports the agent coupled thereto across the blood-brain barrier.

[0377] 23. A method of delivering a therapeutic or diagnostic agent across the blood-brain barrier (BBB) of a subject in need thereof, comprising administering to the subject a complex comprising a therapeutic or diagnostic agent coupled, preferably covalently conjugated, to the antibody or antigen-binding fragment thereof of any one of embodiments 1-5.

[0378] 24. A method of inducing antibody-dependent phagocytosis (ADP) without stimulating the secretion of pro-inflammatory cytokines in a subject in need thereof, comprising administering to said subject a complex comprising a therapeutic antibody or antigen binding fragment thereof coupled, preferably covalently conjugated, to any one of embodiments 1-5, wherein said therapeutic antibody or antigen binding fragment thereof comprises one or more mutations in the Fc domain that reduce or eliminate effector function, such as antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0379] 24a. The method of embodiment 23, wherein said therapeutic antibody or antigen binding fragment thereof comprises one or more amino acid modifications of positions L234, L235, D270, N297, E318, K320, K322, P331 and P329, wherein the numbering of amino acid residues is according to the EU index as shown in Kabat.

[0380] 24b. The method of embodiment 24a, wherein said therapeutic antibody or antigen binding fragment thereof comprises one, two or three mutations of L234A, L235A and P331S.

[0381] 25. The method of any one of embodiments 20-24b, wherein said subject is in need of treatment of a neurological disorder, preferably said neurological disorder is selected from the group consisting of neurodegenerative diseases (e.g. Lewy body disease, post-polio syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, spinocerebellar ataxia, spinal muscular atrophy), tauopathies (e.g. Alzheimer's disease and supranuclear palsy), prion diseases (e.g. bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease and fatal familial insomnia), bulbar palsy, motor neuron disease and neuroaxonal dystrophic diseases (e.g. Canavan disease, Huntington's disease, neuronal ceroid-lipofuscinosis, Alexander disease, Tourette syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Hallervorden-Spatz syndrome, Lafora disease, Rett syndrome, Wilson's disease, Lesch-Nyhan syndrome and Unverricht-Lundborg syndrome), dementia (e.g. Pick's disease and spinocerebellar ataxia) and cancer of the CNS and / or brain (e.g. brain metastases from cancer elsewhere in the body).

[0382] 26. The method of any one of embodiments 20-25, wherein said antibody or antigen binding fragment thereof, complex, multispecific antibody, fusion construct or pharmaceutical composition is administered intravenously.

[0383] 27. The method of any one of embodiments 21-26, wherein the therapeutic agent or therapeutic antibody or antigen-binding fragment thereof is a neurological disorder drug.

[0384] 28. The method of any one of embodiments 21-23, wherein the agent is an imaging agent or an agent for detecting a neurological disorder.

[0385] 29. The method of any one of embodiments 20-28, wherein the anti-TfR antibody or antigen-binding fragment thereof, complex or fusion thereof does not impair binding of TfR to its natural ligand, transferrin.

[0386] 30. The method of any one of embodiments 20-29, wherein the administration reduces Fc- mediated effector functions.

[0387] 31. The method of any one of embodiments 21-30, wherein the administration does not induce rapid reticulocyte depletion.

[0388] 32. The method of embodiment 31, wherein the therapeutic antibody or antigen-binding fragment thereof specifically binds to tau aggregates.

[0389] 33. The method of any one of embodiments 20-32, wherein the subject is a primate, such as a human, more preferably a human having a neurological disorder.

[0390] 34. The method of embodiment 33, wherein the neurological disorder is selected from the group consisting of Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman syndrome, Rett syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, and traumatic brain injury.

[0391] The following examples of the present application further illustrate the nature of the present application. It is understood that the following examples do not limit the present application, and that the scope of the present application is determined by the appended claims. Examples

[0392] Example 1

[0393] While the blood-brain barrier (BBB) prevents harmful substances from entering the brain and is essential for brain homeostasis, it presents a formidable barrier for effective drug delivery to the brain. To this end, a monoclonal antibody (mAb) brain shuttle vector platform was developed that crosses the BBB and results in significantly higher brain concentrations than the mAb alone.

[0394] Antibody generation (OMT rats and Ablexis mice)

[0395] OMT rats (OmniRat®, from Ligand Pharmaceuticals) and Ablexis mice (Ablexis, LLC, San Diego, CA) were immunized with human (SEQ ID NO: 1), cynomolgus (SEQ ID NO: 2) and marmoset (SEQ ID NO: 3) transferrin receptor (TfR) using a repeated immunization multiple sites (RIMMS) protocol for 46 days (Ablexis), 49 days (OMT) or 50 days (OMT). Briefly, animals were repeatedly immunized at multiple subcutaneous sites proximal to regional draining lymph nodes. Serum titrations (ELISA, enzyme-linked immunosorbent assay) were performed at day 32 (OMT) or day 35 (Ablexis) and all animals showed low to moderate titers to human, cynomolgus and marmoset TfR and no titers to negative controls. Lymph nodes were harvested from serum positive rats and mice and fused to produce hybridomas.

[0396] Hybridomas were first screened by Meso Scale Discovery (MSD) or ELISA for binding to HEK293T huTfR (human transferrin receptor) expressing cells. All of these hits were then tested in a confirmatory screen. In the fluorescence-activated cell sorting (FACS)-based confirmatory screen, MDCK-huTfR cells (Madin-Darby canine kidney cells) and pBECs (microvascular endothelial cells, endogenous huTfR expression) were used and MDCK (parental) cells were used as a negative cell line. After the confirmatory screen, 616 TfR-specific cell binders were identified (bind to either or both huTfR expressing cells). From these 616 hits, 340 bound on pBECS and MDCK-huTfR cells, 16 bound only on pBECS and 260 bound only on MDCK-huTfR.

[0397] Hybridomas that bound to pBECs and MDCK-huTfR cells were then screened for binding to rat TfR (SEQ ID NO: 4) and mouse TfR (SEQ ID NO: 5), checked for internalization in pBECs and competition with TfR. RNA lysates were prepared for those mAbs that had human, cynomolgus and marmoset cross-reactivity and internalized without competing for TfR. Antibody V-region sequencing data was obtained.

[0398] Antibody generation (llamas)

[0399] To generate a single domain (VHH) antibody against human TfR that is cross-reactive with cynomolgus monkey, mouse and rat, two llamas were immunized at Abcore in project 452L (animals 1663L and 1663L). Antibody titers were determined by ELISA using TfR protein (1 pg / ml). Three bleeds from both animals were tested and both animals showed good early titers.

[0400] Phage display was performed at Abcore using their standard protocol. Two libraries were made: library 1 (452L-1) from the second bleed of both animals, and library 2 (452L-2) from the second and third bleeds. Plasmid DNA from 12 random individual clones was sequenced and >80% contained VHH inserts with correct reading frame. Both phage display libraries were screened using standard Abcore panning procedure with human TfR. Three rounds of panning were performed with 10 pg / ml human TfR. After panning, 94 individual clones were screened by phage ELISA for specific binding to protease-activated receptor 1 (Parl) N-terminal domain and non-specific binding to BSA (bovine serum albumin). Cross-reactivity with cynomolgus monkey, mouse and rat TfR was measured. The 94 clones were selected for sequence analysis.

[0401] Phage antibody generation

[0402] A phage library was panned against biotinylated huTfR complexed with transferrin. The biotinylated complex was captured on streptavidin magnetic beads (Dynal) and exposed to a fresh pIX Fab library pre-incubated with a final concentration of 100 nM (rounds 1 and 2) or 50 nM (rounds 3 and 4) of transferrin protein. Non-specific phage were washed away in PBS-Tween and bound phage were recovered by infecting MC1061F' E. coli cells. Phage were amplified from these cells overnight and the panning was repeated for a total of four rounds. After four rounds of biopanning, monoclonal Fabs were screened in ELISA for binding to human transferrin receptor. Clones that demonstrated binding to transferrin receptor were sequenced in the heavy and light chain variable regions.

[0403] Examples of TfR antibodies or antigen binding fragments of the invention are outlined in Table la below.

[0404] The binding affinity (KD, konor kaand koffor kdis or kd) of the anti-TfR mAbs as part of the tripartite fusion construct (BBBB construct) described in more detail below to TfR at neutral pH (7.4) and acidic pH (5) was measured using the following Biolayer Interferometry method. The results are shown in Table lb below.

[0405]

[0406]

[0407]

[0408] Table 1b

[0409]

[0410]

[0411] Tri-Foot construct design

[0412] Antibodies against TfR were generated by immunizing rodents and llamas. The resulting mAbs were screened for competition with transferrin and non-competitive mAbs in the tri- foot mAb (also called TTP mAb) format and as scFv or Nanobody formats and characterized. Tri- feet are used to deliver substances of interest (e.g., monoclonal antibodies) to the brain. More particularly, tri-foot constructs containing an antigen-binding fragment of an antibody against TfR and a fusion of a monoclonal antibody (mAb) of interest were developed ( Figure 1 ) to help the mAb cross the BBB and result in significantly higher brain concentrations of the mAb than the mAb alone.

[0413] For example, tri-foot mAbs consist of a therapeutic mAb and a TfR-binding scFv or Nanobody attached to the C-terminus of one of the antibody heavy chains using a short, flexible linker. Tri-foot mAbs were analyzed for features previously described as enhancing transcytosis (reviewed in Goulatis and Shusta 2017): valency, binding affinity, pH-dependent binding, and rapid internalization in brain endothelial cells. Figures 2-4

[0414] Heavy and light chain variable sequences of antibodies against TfR were fused as single chain variable fragments (scFv) in a single genetic construct using the following format: Hc_GTEGKSSGSGSESKST (SEQ ID NO: 314)_Lc. scFv or VHH against TfR were then fused to the C-terminus of the heavy chain (Hc) of an antibody of interest using a GGSGGS (SEQ ID NO: 312) or GGAGGA (SEQ ID NO: 313) linker. Zymeworks heterodimerization mutations in CH3 were utilized in the antibody Hc (Hc A: T350V_L351Y_F405A_Y407V; Hc B: T350V_T366L_K392L_T394W) to generate tri-foot constructs ( Figure 1 ​), also known as triabodies. Triabodies contain two light chains with identical amino acid sequences and two heavy chains with different amino acid sequences. Only one of the two heavy chains is fused to the scFv or VHH of the TfR antibody of the application, and the two heavy chains also differ in their constant regions to promote heterodimerization between the two heavy chains. Thus, each triabody according to embodiments of the application is associated with three amino acid sequences: the amino acid sequence of the first heavy chain fused to the antigen binding fragment of the TfR antibody, the amino acid sequence of the light chain, and the amino acid sequence of the second heavy chain not fused to the antigen binding fragment of the TfR antibody.

[0415] Tri-Foot expression and purification

[0416] Triabodies were expressed in CHO-Expi cells and purified using protein A affinity chromatography followed by size exclusion chromatography or ion exchange chromatography.

[0417] Examples of triabodies made are provided in Table 2a:

[0418] Table 2a

[0419]

[0420]

[0421]

[0422]

[0423] Cellular binding and TfR specificity

[0424] Triabodies were analyzed for features previously described as enhancing transcytosis (reviewed in Goulatis and Shusta 2017): valency, binding affinity, pH-dependent binding, and rapid internalization in brain endothelial cells. Figures 2-4

[0425] Human brain endothelial cells (hCMECD3, 50,00 cells) were incubated with 10 ug / mL purified triabodies and incubated overnight at 4°C in the presence or absence of 10x molar concentration of huTfRl ECD (SEQ ID NO: 1). The next morning cells were fixed and washed, incubated with secondary antibody (Jackson Immunosciences Cat# 109-546-170), washed again, and then analyzed by FACS. Positive binders were defined as having greater than 2-fold binding signal over isotype control and a binding signal / ratio of binding signal with TfR ECD of > 2 (Table 2b).

[0426] ​Table 2b: hCMECD3 cell binding and specificity of Tripod mAbs.

[0427]

[0428] Additional hCMECD3 cell binding assays were performed to measure specificity for additional Tripod mAbs, the results of which are shown in Table 2c below:

[0429] Table 2c

[0430]

[0431] Transferrin competition

[0432] MDCK cells expressing recombinant human transferrin receptor were plated at 10,000 cells / well into MA6000 384 HB plates and incubated in DMEM media supplemented with 10% FBS and 500 μg / mL geneticin for 18 hours. Prior to assay, cells were incubated in serum-free DMEM media supplemented with 5 μM monensin at 37°C in a CO2incubator for 1 h, then incubated with StartingBlock (PBS) supplemented with 5 μM monensin for 30 minutes at room temperature. Cells in alternate rows of the plate were incubated with 2.7 mg / mL human holotransferrin prepared in serum-free DMEM media supplemented with 5 μM monensin for 30 minutes at room temperature. Test antibodies were diluted to 5 μg / mL in serum-free DMEM media supplemented with 5 μM monensin and added to replicate wells containing holotransferrin or replicate wells that did not receive transferrin, then incubated for 1 h at room temperature. Supernatant was removed and 2 μg / mL Sulfo-TAG labeled anti-human antibody was added to each well and incubated for 30 minutes at room temperature. All wells were washed with PBS and no-surfactant MSD Read Buffer T was added. Plates were read on an MSD SECTOR® S600 imager.

[0433] Statistical analysis was performed in Excel, including mean, standard deviation, and RSD. Any sample with an RSD > 25% was excluded. The mean of the test antibody incubated in the presence of transferrin was compared to the mean in the absence of transferrin. Antibodies with values in the presence of transferrin < 70% of the values in the absence of transferrin were considered to be competing for the ligand (Table 3).

[0434] Table 3: Selected Tripod mAbs do not compete with transferrin.

[0435] Sample ID SEQ ID NO: BBBB434 10、11、12 1758 1762 100.3 BBBB501 35、36、37 1348 1618 120.0 BBBB509 63、64、65 282 447 158.6 BBBB520 91、92、93 1369 1798 131.3 BBBB534 116、117、118 1350 1141 84.5 BBBB537 141、142、143 1504 1851 123.1 BBBB543 169、170、171 225 233 103.8 BBBB556 197、198、199 1246 1531 122.9 BBBB557 225、226、227 598 530 88.7

[0436] Internalization

[0437] Human cerebral endothelial cells (hCMEC / D3) were plated at 10,000 cells / well into collagen-coated 384-well Cell Carrier Ultra plates (Perkin Elmer) and allowed to adhere for 16 hours at 37°C in a humidified incubator. Cells (50,00 cells) were then incubated with 200 ug / mL purified tripartite mAbs and allowed to incubate for 1 hour at 37°C. Cells were fixed, washed and incubated with fluorescently labeled secondary antibodies for 1 hour. Cells were then washed again and incubated with fluorescently labeled actin stain, phalloidin, and nuclear stain, Hoeschst 33342. Cells were washed again and imaged using an ImageXpress Micro (Molecular Devices) at 40x objective. MetaXpress 6.0 was used to identify internalized mAbs based on co-localization with phalloidin. All mAbs from Tables 2 and 3 were positive for internalization.

[0438] Affinity analysis and pH-dependent binding, species cross-reactivity

[0439] Affinity and pH dependence was initially measured using the Forte Bio Octet platform. Biotinylated huTfR was immobilized on streptavidin sensors and mAb association was performed for 180 seconds in 0.1 M Phosphate pH 7.4. Dissociation was performed for 300 seconds in 0.1 M Phosphate pH 7.4 or 0.1 M Phosphate pH 5 (Table 4). Preferably, tripartite mAbs of interest have high binding affinity at pH 7.4 and low binding affinity at pH 5, for example KD≥ 1 nM and kd≥ 10 -4 sec -1 , preferably about 10 -3 , such that the tripartite mAb binds TfR at neutral pH (e.g., pH 7.4) and dissociates from TfR at acidic pH (e.g., pH 5). Preferably, the KD at acidic pH and neutral pH are similar, for example the ratio of acidic KD / neutral KD is about 1.5.

[0440] Table 4: Kinetic rate constants measured for huTfR using the Octet platform.

[0441]

[0442] To obtain additional accuracy for affinity measurements, the affinities of tripartite mAbs for huTfR were determined using surface plasmon resonance (SPR) on a BioRad Proteon instrument ProteOn XPR36 system. An Fc capture surface was generated by coupling anti-IgG Fc mAb (Jackson ImmunoResearch) to a GLC chip (BioRad) using amine coupling chemistry (BioRad). Tripartite mAbs were captured at a target density of 120 RU using a concentration of 0.3 ug / mL at 60 uL / min for 30 seconds. huTfR was then flowed over immobilized tripartite mAbs at concentrations ranging from 3.125 - 800 nM (4-fold serial dilution) for 3 minutes (50 µL / min) association followed by 50 uL / min dissociation for 10 minutes. The chip surface was regenerated with 2 18-second pulses of 100 mM H3PO4 (Sigma) at 100 µL / min. Collected data were processed using ProteOn Manager software V3.1.0.6 (BioRad). First, data were corrected for background using inter-spots. Then, for analyte injections, a double reference subtraction of data was performed by using buffer injections. Kinetic analysis of data was performed using a Langmuir 1 : 1 binding model. Results for each mAb are reported in the format of Ka (association rate), Kd (dissociation rate), and KD (equilibrium dissociation constant) (Table 5).

[0443] Table 5: Binding affinities of anti-TfR brain shuttle vectors for TfR when fused to B21M mAb or BACE mAb.

[0444]

[0445] pH-dependent binding was evaluated using the SPR (proteon) method described above, with the exception that the buffer pH was stepped from 7.4 to 6.5 to 6.0 during dissociation. If the dissociation rate increased as the pH decreased, each sensorgram was evaluated and scored for pH-dependent binding (Example in Figure 3

[0446] Species cross-reactivity was evaluated using the same method as for determining binding affinity, with the exception that the TfR used was cynomolgus monkey (SEQ ID NO: 2), marmoset (SEQ ID NO: 3), rat (SEQ ID NO: 4), and mouse (SEQ ID NO: 5). No rat or mouse cross-reactive mAbs were identified. Cynomolgus monkey and marmoset cross-reactive tripartite mAbs were identified (Table 6).

[0447] Table 6: Species cross-reactivity for selected tripartite mAbs​

[0448]

[0449] Anti-TfR antibodies or antigen-binding fragments of the application can be used to deliver any type of immunoglobulin. Similar results have been observed with IgGl and IgG4 therapeutic mAbs delivered by the tripod structure (data not shown).

[0450] Mouse pharmacokinetics and pharmacodynamics and anti-BACE mAb brain shuttle vectors

[0451] To analyze the impact of binding properties on transcytosis, in vivo PK / PD studies were performed in mice. C57BL / 6-Tfrctm2618 (TFRC) Arte mice (Taconic Artemis) were dosed with test articles by IV bolus (13 mg / kg, 10 mL / kg). At predetermined time points, mice were anesthetized by inhalation of isoflurane. Blood was collected by cardiac puncture, and plasma was processed. Mouse brains were collected after systemic infusion with 5 mL of a 0.9% saline solution. Collected brain samples (without cerebellum) were divided into right / left hemispheres, snap-frozen in liquid nitrogen, and stored at -70°C until tissue homogenization and capillary depletion processing.

[0452] None of the TfR-binding molecules cross-reacted with murine TfR, so human TfR KI mice were used to evaluate transcytosis. The tripod mAbs were formatted with an anti-β-secretase 1 (BACE1) antagonist mAb to allow pharmacodynamic evaluation of the mAb after transcytosis into the brain. BACE1 cleaves β-amyloid to release Αβ 1-40 . Inhibition of BACE1 was measured by quantifying the product Αβ 1-40 In the brain. Mice were dosed intravenously with two tripod mAbs, BBBB383 and BBBB426, and a control mAb, BBBB456. BBBB456 is a standalone anti-BACE1 antagonist mAb. BBBB383 and BBBB426 differ only in their affinity for TfR, with K D = 18 nM and 130 nM, respectively. After infusion and capillary depletion to reduce interference from mAb in the blood or retained within the vascular endothelium, brain exposure was determined (Johnsen, Burkhart et al. 2017). The brain concentrations of both BBBB383 and BBBB426 were increased at all time points compared with BBBB456, with BBBB383 having greater mAb brain concentrations than BBBB426. A strong PK / PD relationship was observed, with mAb brain concentration correlating with reduction in Αβ 1-40 Lower plasma exposure of the two TfR-containing mAbs was attributed to TMDD by binding TfR in the periphery.

[0453] The selected anti-TfR brain shuttle vector was then fused to a prototype anti-BACE (beta-secretase) mAb and binding affinity was re-evaluated using the same method described above. As shown in Table 5, the affinity of the anti-TfR brain shuttle vector was similar when fused to the B21M mAb (anti-human respiratory syncytial virus) and the anti-BACE antagonist mAb. Internalization was evaluated for the selected molecules and was found to be unchanged from that observed when the anti-TfR brain shuttle vector was fused to the B21M mAb.

[0454] In vivo rodent studies were performed in huTfR knock-in mice (C57BL / 6-Tfrctm2618 (TFRC)Arte mice (Taconic Artemis)) using a prototype anti-BACE antagonist mAb (BBBB456, SEQ ID NOs: 307, 308, and 309) due to the lack of anti-TfR brain shuttle vector binding to mouse or rat TfR. The anti-BACE antagonist mAb was selected as a model PD system for measuring inhibition of BACE1 (by concentration of its product peptide Aβ1-40), which reflects the amount of mAb transported to the brain.

[0455] The first in vivo study evaluated the PK / PD relationship in the brain of huTfR mice. i.v. BBBB383 (SEQ ID NOs: 256, 257, and 258), BBBB426 (SEQ ID NOs: 275, 276, and 277), and BBBB456 (SEQ ID NOs: 307, 308, and 309) were administered to knock-in (KI) mice at 13 mg / kg. Brains and plasma were harvested at 4, 24, and 72 hours. At the predetermined time points, mice were anesthetized by inhalation of isoflurane. Mouse brains from KI mice were collected after systemic infusion of 5 mL of a 0.9% saline solution. The collected brain samples (lacking cerebellum) were divided into right / left hemispheres, snap-frozen in liquid nitrogen, and stored at -70 °C until tissue homogenization and capillary depletion processing.

[0456] For sample preparation of capillary-depleted brain tissue lysates, individual weights were obtained for brain hemispheres to measure drug concentrations. Brain tissue samples were added to a calculated volume of modified dPBS buffer containing protease inhibitors (Pierce; A32955) (2.5-3 μΐ, buffer per 1 mg of tissue) and transferred to Lysing Matrix D (MP Biomedicals™; 6913-100) tubes. Tissue samples were homogenized at 2.9 m / s for 15 seconds using a Bead Ruptor 24 Elite (Omni International). The entire cell suspension was transferred to a new tube and mixed with an equal volume of 26% dextran buffer (13% final dextran concentration). The mixed tissue homogenate was centrifuged at 2,000 g for 10 minutes at 4°C. Carefully, the upper layer (capillary-depleted fraction) was separated from the remaining sample and transferred to a new tube containing 10x RIPA lysis buffer (Millipore™; 20-188). The capillary-depleted sample plus lysis buffer was vortexed thoroughly, centrifuged at 14,000 rpm for 30 minutes at 4°C, and the supernatant was transferred to a new tube. Brain tissue sample lysates were stored frozen at -70°C or measured for protein concentration using a BCA Protein Assay Kit (Pierce™; 23227). Final brain tissue sample lysates were normalized to a total protein concentration of 7 mg / mL prior to immunoassay determination of BBB-permeable mAbs.

[0457] For PK evaluation, the concentration of BBB-effective mAb in mouse brain tissue was determined using MesoScale Discovery (MSD®; Gaithersburg, MD) ECLIA technology developed in a typical sandwich immunoassay format. Assays were performed on MSD Gold™ Small Spot Streptavidin 96 well plates (Cat: L45SA). Briefly, streptavidin-coated plates were blocked with 1% bovine serum albumin (BSA) / lx phosphate buffered saline (PBS) for 30 minutes at room temperature. Freshly made standard curves were prepared by serial dilution in 50% naive C57BL / 6 mouse brain tissue lysate. Frozen quality controls (QCs) prepared in naive C57BL / 6 mouse brain tissue lysate were diluted and tested at 2x working assay concentration with each assay. Master mix containing capture (biotinylated anti-human Fc mAb, 1 μg / mL) and detection (ruthenium-labeled anti-human Fc mAb, 0.5 μg / mL) reagents were combined with diluted standards, QCs, and samples in a 1 : 1 volume ratio in assay plates. The mixtures were incubated for 1 hour at room temperature with shaking. Assay plates were washed, and MSD T-Read Buffer (lx) was added to all wells. Raw data values were read on a MSD SECTOR® S600 imager. The standard curve range for the assay was tested from 1 - 512 ng / mL, with a minimum required sample dilution (MRD) of 1 :2, resulting in a sensitivity limit of 2 ng / mL for brain tissue lysate. MSD output files with raw ECL counts were imported into Watson LIMS (Thermo Scientific), followed by a 5-parameter logistic fit with 1 / F2 weighted regression.

[0458] For PK evaluation, the concentration of BBB-effective mAbs in mouse plasma was determined using a similar protocol as described above. A new standard curve was prepared by serial dilution in 10% pooled mouse plasma. Dilutions and a test of frozen QCs prepared in pooled mouse plasma were tested at 10x working assay concentration with each assay. Master mix containing capture (biotinylated anti-human Fc mAb, 1 μg / mL) and detection (ruthenium-labeled anti-human Fc mAb, 0.5 μg / mL) reagents was combined with diluted standards, QCs, and samples in a 1 : 1 volume ratio in assay plates. The mixture was incubated for 1 hour at room temperature while shaking. The assay plates were washed, and MSD T read buffer (lx) was added to all wells. Raw data values were read on a MSD SECTOR® S600 imager. The standard curve range for the assay was tested from 2 - 512 ng / mL with an MRD of 1 : 10, resulting in a sensitivity limit of 20 ng / mL in plasma matrix. The MSD output file with raw ECL counts was imported into Watson LIMS (Thermo Scientific) and fitted with a 5-parameter logistic fit with a 1 / F2 weighted regression.

[0459] BACE activity measurements were performed by homogenizing mouse brains in 2 ml lysis matrix D tubes (8 μΐ 0.4% DEA / 50 mM NaCl per mg of brain weight, Fast Prep-24, 6 / shake / sec for 20 sec). Tubes were then centrifuged in an Eppendorf Centrifuge set to maximum speed for 5 min at 4°C. The homogenate (supernatant) was then transferred to pre-chilled tubes which were then centrifuged at 13,000 rpm for 70 min at 4°C. The supernatant was then transferred to tubes containing 10% 0.5 M Tris / HCL and frozen at -80°C until assay. Aβ 1-40 peptide standards and thawed treated brain homogenate were pre-complexed with ruthenium (Meso Scale Discovery (MSD), R91AN-1) labeled anti-Aβ antibody at 1 : 1. 50 ul of the complex was added to a blocked plate containing Aβ 1-40 capture antibody. After overnight incubation at 2-8°C without shaking, the plate was washed and 2x read buffer (MSD, R92TC-1) was added. The plate was read using a Meso Sector S 600 (MSD, IC0AA).

[0460] It was observed that mAbs BBBB383 and BBBB426 containing brain shuttle vectors had a faster plasma clearance than anti-BACE mAb BBBB456 alone Figure 5A). However, in the brain, BBBB383 and BBBB426 were observed to have increased brain concentrations compared to control BBBB456 at all time points. When measuring the PD effect of BACE inhibition, both brain shuttle mAbs were observed to inhibit BACE activity to a greater extent than the control anti-BACE mAb alone Figure 5B

[0461] At 13 mg / kg i.v. At 4 and 24 hours post-dose, additional brain shuttle containing mAbs were similarly evaluated Figure 7A -B). Similar to the first study, all brain shuttle mAbs were observed to have faster plasma clearance than the control anti-BACE mAb. A range of brain concentrations were observed for the brain shuttle mAbs, with increased brain concentrations for all but BBBB974. It is hypothesized that BBBB974 is not efficiently transported to the brain due to its binding kinetics. In particular, BBBB974 has a slow on-rate which can prevent efficient association with TfR in vivo. A tripod mAb concentration dependent decrease in Αβ1-40 levels was also observed for all tripod mAbs except BBBB983, which had increased brain concentrations compared to control BBBB456, but did not affect Αβ1-40 concentrations Figure 8 ). This observation can be attributed to binding kinetics, as BBBB983 has a very slow off-rate which can prevent efficient diffusion in the brain, which is necessary for BACE inhibition. These data highlight the importance of TfR binding kinetics for both delivery and function of therapeutic mAbs.

[0462] The relationship between affinity and transcytosis efficiency has been previously described as transcytosis increasing with decreasing affinity for TfR (Yu, Zhang et al. 2011), which is inconsistent with the data above. To probe the transcytosis affinity relationship in more detail, brain PK / PD of 9 tripod mAbs were evaluated in the mouse model described above. These tripod mAbs varied by approximately 100-fold in affinity for TfR (K D Brain concentrations (C max脑 ) were measured 24 hours post-IV dose (Figure 17). As expected, a range of transcytosis efficiencies were observed, ranging from no enhancement to a 10-fold increase compared to the control mAb. This data suggests a more nuanced relationship between affinity and transcytosis efficiency than previously described, where the contributions from both on- and off-rates impact brain concentrations. For example, no enhancement in brain exposure was observed for BBBB946 compared to the control mAb, despite its K D ​= 65 nM and a fast off-rate. This mAb is unique, but has a slower on-rate (k a ≈ 10 3 M −1 s −1 , compared to k a ≈ 10 5 M −1 s −1 ). In fact, the contrast is striking when compared to another tripedal antibody, BBBB969, which has a similar K D (K D = 81 nM) but a 100x faster on-rate. BBBB969 enhances brain concentration by 5.5-fold, demonstrating the importance of a fast enough on-rate for effective brain delivery. The efficiency of transcytosis for the other 8 mAbs studied can best be described by their off-rates, with optimal brain delivery occurring at off-rates that are neither too fast nor too slow (2 x 10 -3 s -1 , the optimal neutral k d ). A strong PK / PD relationship was observed for all tripedal mAbs except BBBB983, which has a 5.5x brain concentration enhancement but does not affect Αβ 1-40 levels. This mAb has a slow neutral off-rate (< 8 x 10 -5 s -1 ), which we hypothesize impacts its ability to diffuse to the target in the brain. In summary, the data demonstrate the importance of optimizing both the neutral on- and off-rates for optimal brain PK and PD. We did not observe an effect of the binding epitope to TfR in the study (data not shown).

[0463] Selection of mAbs for cynomolgus monkey studies and evaluation of brain shuttle vectors fused to anti-Tau mAbs

[0464] Confirmation of the ability of tripedal mAbs targeting TfR in humans to enhance therapeutic antibody brain exposure is key in non-human primates. The tripedal mAbs that performed best in the mouse study (BBBB979 and BBBB978) do not bind cynomolgus TfR and were thus excluded from further study. The next best, BBBB970 and BBBB969, both contain free cysteine residues in the light chain of the anti-TfR brain shuttle (SEQ ID NO: 162 and SEQ ID NO: 218). Since free cysteine residues can contribute to non-ideal biophysical properties during manufacturing, the free cysteine was mutated to a serine residue (SEQ ID NO: 278 and SEQ ID NO: 291).

[0465] New scFv were fused to anti-Tau mAb PT1B844 (SEQ ID NO: 310 and 311) to generate BBBB1136 (SEQ ID NO: 285, 286 and 287) / BBBB1134 (SEQ ID NO: 288, 289 and 290), and BBBB1133 (SEQ ID NO: 298, 299 and 300) / BBBB1131 (SEQ ID NO: 301, 302 and 303) (IgGl AAS YTE / IgGl). Affinity for huTfR was measured (Table 7).

[0466] Table 7: Binding affinity of anti-TfR brain shuttle vectors fused to anti-Tau mAbs for huTfR with Cys-Ser mutations

[0467]

[0468] BBBB1134 / BBBB1136 maintained very similar binding to huTfR as BBBB557 / BBBB970, indicating that neither the Cys-Ser mutation nor fusion to anti-Tau mAb interfered with binding affinity for huTfR. However, the binding affinity of BBBB1131 / BBBB1133 was about ½ of that of BBBB543 / BBBB969. To determine if the change in affinity was due to the cys-ser mutation or fusion to anti-Tau mAb, brain shuttle vectors were generated without the mutation but fused to anti-Tau and evaluated for binding (BBBB1048 (SEQ ID NO: 178, 179 and 180) / BBBB1046 (SEQ ID NO: 181, 182 and 183)). The affinity of unmutated BBBB1048 / BBBB1046 was very similar to BBBB543 / BBBB969, indicating that the loss of affinity was due to the cys-ser mutation and not due to fusion to Tau mAb (Table 8).

[0469] Table 8: Binding affinity of anti-TfR brain shuttle vectors fused to anti-Tau mAbs for huTfR

[0470]

[0471] Similar to previous studies, internalization was also evaluated, and fusion of brain shuttle vectors to anti-Tau mAb did not affect their ability to internalize in human brain endothelial cells (Table 9). Figure 9 Examples in Table 9, the mAbs tested.

[0472] Table 9: Evaluation of anti-TfR brain shuttle vectors for internalization in human brain endothelial cells.

[0473] mAb Internalization BBBB1046 Yes BBBB1047 Yes BBBB1048 Yes BBBB1052 Yes BBBB1053 Yes BBBB1054 Yes BBBB1055 is BBBB1131 is BBBB1132 is BBBB1133 is BBBB1134 is BBBB1135 is BBBB1136 is

[0474] Pharmacokinetics of anti-Tau brain shuttling vector mAbs in cynomolgus monkeys

[0475] Test articles were administered to cynomolgus monkeys via IV injection (slow bolus) at the indicated dose. At the predetermined time points, cynomolgus monkey brains were collected and rinsed with cold saline solution after a minimum of 5 minutes of saline body perfusion. The predetermined brain locations were isolated, snap-frozen in liquid nitrogen and stored at -80°C until tissue homogenization and capillary depletion processing.

[0476] In cynomolgus monkeys, BBB1133, BBB1136 and BBB1134 were administered at 10 mg / kg IV i.v. BBB1133, BBB1136 and BBB1134 were administered with the BBB- effective mAbs PT1B844 (Figure 18) and PT1B916. Plasma was sampled at 4, 24 and 72 hours. Cynomolgus monkey brains were collected and rinsed with cold saline solution after a minimum of 5 minutes of saline body perfusion. The predetermined brain locations were isolated, snap-frozen in liquid nitrogen and stored at -80°C until tissue homogenization and capillary depletion processing.

[0477] For sample preparation of capillary-depleted brain tissue lysates, the individual tissue weights of the collected brain locations were obtained. Brain tissue samples were added to a calculated volume of modified dPBS buffer containing protease inhibitors (Pierce; A32955) (2.5 μΐ, buffer per 1 mg tissue) and transferred to Lysing Matrix D (MP Biomedicals™; 6913-100) tubes. Using a Bead Ruptor 24 Elite (Omni International), the tissue samples were homogenized at 2.9 m / s for 15 seconds. The entire cell suspension was transferred to a new tube and mixed with an equal volume of 26% dextran buffer (13% final dextran concentration). The mixed tissue homogenate was centrifuged at 2,000 g for 10 minutes at 4°C. Carefully, the supernatant (capillary-depleted fraction) was separated from the remaining sample and transferred to a new tube containing 10x RIPA lysis buffer (Millipore™; 20-188). The capillary-depleted sample plus lysis buffer was vortexed thoroughly, centrifuged at 14,000 rpm for 30 minutes at 4°C, and the supernatant was transferred to a new tube. Brain tissue sample lysates were stored frozen at -70°C or protein concentration was measured using a BCA Protein Assay Kit (Pierce™; 23227). Final brain tissue sample lysates were normalized to a total protein concentration of 7 mg / mL prior to immunoassay determination of BBB-effective mAbs.

[0478] For PK evaluation, concentrations of BBB-effective mAbs in cynomolgus brain tissue were determined using MSD® ECLIA technology developed in a typical sandwich immunoassay format. Assays were performed on MSD Gold™ Small Spot Streptavidin 96-well plates. Streptavidin-coated plates were blocked with 1% bovine serum albumin (BSA) / lx phosphate-buffered saline (PBS) for 30 minutes at room temperature. New standard curves were prepared by serial dilution in 50% naive cynomolgus monkey brain tissue lysate. Frozen QCs prepared in naive cynomolgus monkey brain tissue lysate were diluted and tested at 2x working assay concentration with each assay. Master mix containing capture (biotinylated anti-human Fc mAb, 1 μg / mL) and detection (ruthenium-labeled anti-human Fc mAb, 0.5 μg / mL) reagents were combined with diluted standards, QCs, and samples in a 1 : 1 volume ratio in assay plates. The mixtures were incubated for 1 hour at room temperature with shaking. The assay plates were washed, and MSD T-Read Buffer (lx) was added to all wells. Raw data values were read on a MSD SECTOR® S600 imager. The standard curve range for the assay was tested from 1 - 512 ng / mL, with a minimum required sample dilution (MRD) of 1 :2, resulting in a sensitivity limit of 2 ng / mL for brain tissue lysate. The MSD output file with raw ECL counts was imported into Watson LIMS (Thermo Scientific), followed by a 5-parameter logistic fit with 1 / F2 weighted regression.

[0479] For pharmacokinetic (PK) evaluation, the concentration of BBB-effective mAbs in cynomolgus monkey plasma was determined using the MSD® ECLIA technique, developed in a typical sandwich immunoassay format. Assays were performed on MSD Gold™ Streptavidin 96-well plates. Streptavidin-coated plates were blocked for 30 min at room temperature with 1% bovine serum albumin (BSA) + 0.5% Tween-20 / 1x phosphate-buffered saline (PBS). New standard curves were prepared in 10% pooled cynomolgus monkey plasma via serial dilutions. Frozen QCs were prepared in pooled cynomolgus monkey plasma at 10x working assay concentrations with each assay dilution and test. A master mixture containing capture (biotinylated anti-human Fc mAb, 1 μg / mL) and detection (ruthenium-labeled anti-human Fc mAb, 1 μg / mL) reagents was combined with diluted standards, QCs, and samples at a 1:1 volume ratio in the assay plate. The mixture was incubated at room temperature for 1 h with shaking. The assay plate was washed, and MSD T-reading buffer (1x) was added to all wells. Raw data values ​​were read out on an MSD SECTOR® S600 imager. Standard curves were tested for the range of 2–512 ng / mL, with a minimum required sample dilution (MRD) of 1:10, yielding a sensitivity limit of 20 ng / mL in the plasma matrix. The MSD output file with raw ECL counts was imported into Watson LIMS (Thermo Scientific) and then fitted with a 5-parameter logistic regression and a 1 / y² weighted regression.

[0480] For mAb, brain concentration was measured across various regions ( Figure 10 Brain concentration data were averaged across animals, and each symbol represents a brain region. Compared to the control mAb, 7×, 11×, and 11× brain concentrations were observed for BBBB1134, BBBB1136, and BBBB1133, respectively. Compared to mAbs without the brain shuttle carrier, all mAbs containing the brain shuttle carrier showed increased brain exposure in each brain region. Figure 11 ).

[0481] The concentration of mAb in plasma was also measured. Figure 12). Evidence for TMDD was observed in the periphery, where the tripod mAb had an accelerated clearance rate compared to the control mAb (Figure 18). The impact of binding to the neonatal Fc receptor (FcRn) was evaluated in this study, where BBBB1134 and BBBB1136 were identical except that BBBB1136 had a "YTE" mutation in the Fc domain (Dall'Acqua, K, et al. 2006). The "YTE" mutation enhances binding to FcRn at acidic pH and has been shown to increase the half-life of mAbs in multiple species, including humans (Robbie, C, et al. 2013). As expected, the addition of the "YTE" mutation resulted in increased plasma concentrations for BBBB1136 compared to BBBB1134. Although FcRn is a key receptor in maintaining IgG homeostasis and extending IgG serum half-life in humans (Roopenian and Akilesh 2007), it is also implicated as a receptor for transcytosis or efflux from the brain (Cooper, C, et al. 2013). We were interested in understanding the interplay between these two functions for FcRn, as improving half-life by increasing binding affinity to FcRn can come at the expense of brain exposure, with increased efflux from the brain. Interestingly, the 2-fold increase in plasma concentration was mirrored by a 2-fold increase in brain concentration, suggesting that any possible increased efflux was negligible in this system.

[0482] BBBB1133 has a peripheral half-life very similar to mAbs PT1B844 and PT1B916, which do not have a brain shuttle vector.

[0483] Reticulocyte depletion in cynomolgus monkeys

[0484] A known negative consequence of TfR targeting to enhance brain exposure is reticulocyte depletion due to antibody-dependent cell-mediated cytotoxicity (ADCC) of reticulocytes in an Fc-dependent manner Science Translational Medicine 2013: Vol. 5, 183). In a cynomolgus monkey PK study for reticulocyte depletion, mAbs were tested with WT IgGl (BBBB1134) and the "AAS" mutation that reduces FcyR binding (BBBB1136 and BBBB1133). As expected, rapid reticulocyte depletion was observed for the WT IgGl tripod mAb BBBB1134, but not for BBBB1136, BBBB1133, or the non-brain shuttle vector mAbs PT1B844 and PT1B916 Figure 13 ), confirming the impact of Fc function on TfR binding mAbs and reticulocyte depletion.

[0485] The third type of three-legged mAb, BBBB1133, was selected for dose-response and repeated-dose PK in cynomolgus monkeys. Cynomolgus monkeys were administered intravenously at doses of 2, 10, and 30 mg / kg, and brain PK was measured at 48 hours, 7, and 14 days later. Plasma PK was evaluated within two weeks. Figure 18A (and B). Linear brain PK was observed between 2 and 10 mg / kg, and nonlinear brain PK was observed between 10 and 30 mg / kg. The proposed delivery mechanism is receptor-mediated, which is saturable, and data indicate that 30 mg / kg is a saturable dose in cynomolgus monkeys. Linear PK was observed in plasma and CSF, with a half-life of approximately 6 days. Repeated dosing was also performed using the same dose range administered weekly for three weeks. Figure 18C (and D). Cumulative evidence of repeated administration of 30 mg / kg was observed, consistent with previous observations that 30 mg / kg was the saturation dose. Linear PK was again observed in the peripheral zone, with no evidence of PK tolerance upon repeated administration.

[0486] Reticulocyte data suggest that effector-silencing Fc mAbs are essential for safe administration via this brain delivery platform. While avoiding reticulocyte depletion is an important safety feature for therapeutic mAbs, this requirement would preclude the use of anti-TfR-mediated brain delivery for any therapeutic mAb that requires effector functionality such as ADP for its therapeutic mechanism of action. For example, one possible therapeutic mechanism of action depends on Fc-dependent microglial phagocytosis of Tau aggregates. By inhibiting the ability of brain shuttle mAbs to bind FcγR to prevent reticulocyte depletion, the mAb cannot bind FcγR on microglia to promote phagocytosis of Tau aggregates.

[0487] To investigate alternative pathways for ADP, we evaluated the ability of effector-silencing triploid mAbs BBBB1133 and BBBB1136 to induce phagocytosis of Tau oligomers in human IPSC-derived microglia. Both triploid mAbs induced greater phagocytosis of Tau oligomers than the control anti-Tau mAb PT1B844 (an IgG1 mAb). Figure 19A It has been confirmed that ADP via BBBB1133 is internalized through TfR-mediated processes and that this process can be blocked by the addition of an excess of soluble TfR extracellular domains. The addition of an excess of soluble Fc does not affect ADP, confirming that non-canonical ADP utilizes TfR rather than FcγR. Figure 19B Tau intracellular transport was observed in BBBB1133 similar to that of the control mAb (PT1B844) via early endosomes (EEA1) to intermediate endosomes (Rab17) and final lysosomes (LAMP1). Figure 19C ).

[0488] To further confirm that non-canonical ADP is a physiologically relevant mechanism for tau degradation by microglia, we evaluated the ability of the tripartite mAbs to induce phagocytosis of human post-mortem Alzheimer's disease-derived tau fibrils (PHF-Tau). ADP of PHF-Tau was measured in human monocyte-derived macrophages and human IPSC-derived microglia (Figure 20). Both PT1B844 and BBBB1133 induced phagocytosis of PHF-Tau at early time points. However, at late time points, BBBB1133 continued to induce ADP of PHF-Tau, while PT1B844-mediated ADP ceased. This can demonstrate that macrophages and microglia exhaust, as described for canonical ADCP (Church, Van Der Meid et al. 2016), and the potential benefit of the non-canonical ADP mechanism utilized by BBBB1133. Similar to the previously described experiments using tau oligomers, uptake of tau was blocked by the addition of excess soluble TfR, confirming that this is a TfR-dependent mechanism. To probe another potential benefit of non-canonical ADP over canonical ADP, pro-inflammatory cytokines were measured in the PHF Tau phagocytosis experiments. As expected, canonical ADP mediated by PT1B844 resulted in secretion of pro-inflammatory cytokines, while non-canonical ADP mediated by BBBB1133 did not.

[0489] To evaluate the potential of AAS IgGl tripartite mAbs to promote uptake of Tau aggregates in microglia, human microglia derived from induced pluripotent stem cells (iPSC) were plated at a dilution of 7000 cells / well onto 384-well PerkinElmer Cell Carrier Ultra plates and maintained in Advanced DMEM / F12 medium with Glutamax+, penicillin / streptomycin, IL34 (100 ng / ml) and GMCSF (10 ng / ml). On the day of the assay, biotinylated phospho-tau oligomers [sequence: SCBiot-(dPEG4)GTPGSRSR(pT)PSLP(pT)PPTREPLL (SEQ ID NO: 315)-amide] were complexed with streptavidin Alexa Fluor 488 (AF488) in a 15-fold molar excess. The labeled phospho-tau oligomers were then allowed to bind to the test mAbs in approximately 2X molar excess for 30 minutes at room temperature. The mAb:tau oligomer complexes were then delivered to the microglia at 20 μΐ / well. After 2, 4 and 8 hours of incubation, the cells were washed twice with phosphate buffered saline (PBS) and fixed in the presence of 4% paraformaldehyde for 15 minutes at room temperature. After fixation, the cells were washed again twice in PBS and incubated overnight at 4°C with LAMP1 primary antibody (marker of lysosomes) at a concentration of 4 μg / ml in permeabilization buffer (0.1% saponin + 1% fish skin gelatin). After incubation, the cells were washed twice with PBS and stained with 1 μg / ml secondary antibody conjugated to Alexa Fluor 647 in permeabilization buffer for 1 hour at 4°C. After incubation, the cells were washed twice with PBS, counterstained with 1 μg / ml Hoechst DNA stain in PBS for 10 minutes at room temperature. The cells were then washed a final time in PBS, resuspended in 20 μΐ PBS / well, and imaged on an Opera Phenix confocal high content microscope. The acquired images were analyzed using Harmony and ImageJ analysis software. Approximately 500 cells / condition were scored for the presence of tau oligomers within phagolysosomal structures and labeled with LAMP1 antibody.

[0490] All brain shuttle mAbs promoted more efficient uptake into phagosomes than non-brain shuttle mAb PT1B844 Figure 15). Within the brain shuttle mAbs, those with full effector function (BBBB 1131, 1134, and 1046) were more effective than those without effector function. These data demonstrate that abrogating binding to FcyRs to reduce the risk of reticulocyte depletion should not impact the therapeutic efficacy of anti-Tau mAbs. Indeed, TfR-mediated internalization and trafficking to phagolysosomes appears to be more efficient in microglia than traditional FcyR-mediated phagocytosis.

[0491] To investigate whether this observation could be repeated using other targets and cells, uptake of RSV F-protein was evaluated in human macrophages. Primary human macrophages were plated at a dilution of approximately 6000 cells / well onto 384-well PerkinElmer Cell Carrier Ultra plates and cultured in X-VIVO 10 serum-free hematopoietic cell medium supplemented with 10% FBS, 50 mg / ml macrophage colony-stimulating factor (mCSF) CSF, and 25 ng / ml interferon gamma (IFNy). On the day of the assay, an approximately 7-fold molar excess of RSV-F protein (His-tagged F protein complexed with anti-His biotinylated antibody and streptavidin Alexa Fluor 488) was bound to anti-RSV mAb (1 ug / ml) for 30 minutes at room temperature. The mAb:F protein complex was then delivered to the macrophages at 20 ul / well. Alexa Fluor 488-labeled E. coli was used as a positive control for phagocytosis. Three hours post-incubation, cells were washed twice with phosphate-buffered saline (PBS) and fixed in the presence of 4% paraformaldehyde for 15 minutes at room temperature. Following fixation, cells were washed twice more in PBS and incubated overnight at 4°C in permeabilization buffer (0.1% saponin + 1% fish skin gelatin) with LAMP1 primary antibody (marker for lysosomes) at a concentration of 4 ug / ml. Following incubation, cells were washed twice with PBS and stained with 1 ug / ml secondary antibody conjugated to Alexa Fluor 647 for 1 hour at 4°C in permeabilization buffer. Following incubation, cells were washed twice with PBS, counterstained with 1 ug / ml Hoechst DNA dye for 10 minutes at room temperature in PBS. Cells were then washed a final time in PBS, resuspended in 20 ul PBS / well, and imaged in an OperaPhenix confocal high-content microscope. The resulting images were analyzed using Harmony and ImageJ analysis software. Approximately 300 cells / condition were scored for the presence of F protein foci within the phagolysosomal structures labeled with LAMP1 antibody.

[0492] As observed for Tau and microglia, all brain shuttle mAbs promoted more efficient uptake into phagosomes than the non-brain shuttle mAb B21M-IgG1 (Figure 16 ). However, no difference in uptake was observed between the IgGl (BBBB932 and BBBB934) and IgGl AAS (BBBB354 and BBBB368) brain shuttle mAbs. The difference between the B21M experiment and the Tau experiment ( Figure 15 and Figure 16 ) is attributed to the target or the cell remains to be determined. However, the data confirm the robustness of the mechanism where TfR-mediated internalization and trafficking to the phagolysosome appears to be at least as efficient as the classical FcyR-mediated phagocytosis.

[0493] To the best of the inventors' knowledge, no publication describes the utilization of this non-canonical ADP mechanism for therapeutic mAbs. While not wishing to be bound by theory, it is believed that both phagocytosis and endocytosis can lead to degradation by the converging phagolysosomal pathway, such that regardless of the internalization trigger (FcyR-mediated phagocytosis or TfR-mediated endocytosis), the internalized cargo is trafficked to the phagolysosome and degraded by it.

[0494] Evaluation of PK / PD relationship in the retina of huTfR mice

[0495] The selected anti-TfR brain shuttle was then fused to a prototype anti-BACE (β- secretase) mAb and binding affinity was re-evaluated using the same method described above. As shown in Table 5, the affinity of the anti-TfR brain shuttle was similar when fused to the B21M mAb (anti-human respiratory syncytial virus) and the anti-BACE antagonist mAb. For the selected molecules, internalization was evaluated ( Figure 4 ) and found to be unchanged from the internalization observed when the anti-TfR brain shuttle was fused to the B21M mAb.

[0496] Since none of the anti-TfR brain shuttles bind to mouse or rat TfR, in vivo rodent studies were performed in huTfR knock-in mice (C57BL / 6-Tfrctm2618(TFRC)Arte mice (Taconic Artemis)) using a prototype anti-BACE antagonist mAb (BBBB970, BBBB978, BBBB983). The anti-BACE antagonist mAb was chosen as a model PD system to measure inhibition of BACE1 (by concentration of its product peptide Αβ1-40), which reflects the amount of mAb trafficked to the brain.

[0497] The first in vivo study evaluated the PK / PD relationship in the retina of the huTfR mice. i.v.Knock-in (KI) mice were dosed with 10 mg / kg of BBBB970, BBBB978, BBBB983 and control BBBB456. Eyes and plasma were harvested at 4 and 24 hours post-dose. At the predetermined time point, mice were anesthetized by inhalation of isoflurane. Mouse eyes from KI mice were collected after systemic infusion of 5 mL of a 0.9% saline solution. Collected eye samples (minus optic nerve) were snap-frozen in liquid nitrogen and stored at -70°C until tissue homogenization or prepared for immunohistochemistry.

[0498] BACE activity measurements were performed by homogenizing mouse eyes in lysis matrix D tubes (8 μl 0.4% DEA / 50 mM NaCl per mg of brain weight, Fast Prep-24, 6 / shake / sec for 20 sec). Tubes were then centrifuged in an Eppendorf Centrifuge set to maximum speed for 5 min at 4°C. The homogenate (supernatant) was then transferred to pre-chilled tubes which were then centrifuged at 13,000 rpm for 70 min at 4°C. The supernatant was then transferred to tubes containing 10% 0.5 M Tris / HCL and frozen at -80°C until assay. Aβ 1-40 peptide standards and thawed eye homogenates were then pre-complexed with ruthenium (MesoScale Discovery (MSD), R91AN-1) labeled anti-Aβ antibodies at 1:1. 50 ul of the complex was added to blocked plates containing capture antibodies for Aβ 1-40. After overnight incubation at 2-8°C without shaking, plates were washed and 2x reading buffer (MSD, R92TC-1) was added. Plates were read using a Meso Sector S 600 (MSD, IC0AA).

[0499] Cytokine secretion analysis

[0500] The relative concentration of secreted proteins in the cell supernatant was measured using an antibody-based 29-plex immunoassay (Luminex, R&D systems, Cat. # LXSAHM-29) after different treatments on human iPSC-derived microglia. The 29 secreted proteins are: BDNF, CCL3 / MIP1a, CCL20 / MIP3a, GroB / MIP2, CXCL10 / IP10 / CRG2, GCSF, IFNa, IL1a, IL2, IL6, IL10, IL17 / IL17a, MCSF, RAGE / AGER, TNFa, CCL2 / JE / MCP1, CCL4 / MIP1b, CXCL9 / MIG, FGFb / FGF2, GMCSF, IFNy, IL1b, IL4, IL8 / CXCL8, IL12p70, IL23, MMP9, Resistin.

[0501] PHF Tau

[0502] Post-mortem cortical tissue obtained from 5 histologically confirmed AD patients (Braak stages V-VI) was used to generate partially purified PHF-tau pools by a modified method of (Mercken et al, Acta Neuropathologica (1992) 84:265-272; Greenberg et al., J. biol. Chem. (1992) 267: 564-569). Typically, 5 g of parietal or frontal cortex was homogenized in 10 volumes of cold buffer H (10 mM Tris, 800 mM NaCl, 1 mM EGTA and 10% sucrose / pH 7.4) using a glass / Teflon Potter tissue homogenizer (IKA Works, Inc; Staufen, Germany) at 1000 rpm. The homogenized material was centrifuged at 27000 x g for 20 min at 4°C. The pellet was discarded and the supernatant was adjusted to a final concentration of 1% (w / v) N-lauroylsarcosine and incubated at 37°C for 2 h. Subsequently, the supernatant was centrifuged at 184000 x g for 90 min at 20°C. The pellet was carefully washed in PBS and resuspended in 750 uL PBS, aliquoted and frozen at -80°C. The quality of the PHF-tau preparations was evaluated by using the AT8 / AT8 phospho-aggregate selective MSD ELISA. Tau content was determined by Western blot using hTau10 (Janssen R&D) with recombinant 2N4R Tau as calibrator.

[0503] Investigation of the ability of TfR TTP mAb to enhance ADP in vivo

[0504] The TfR TTP mAb's ability to enhance ADP in vivo was investigated in a tau- inoculated mouse model. The mouse model used transgenic Tau-P301L mice expressing the longest human tau isoform with the P301L mutation (tau-4R / 2N-P301L) (Terwel et al. (2005) J Biol Chem; 280(5): 3963-73). Due to the lack of TfR cross-reactivity in mice TTP, a mouse surrogate TTP with similar binding properties to the primary human TfR TTP was developed and used in this study. The tau-inoculated model involves stereotactic hippocampal injection of PHF-Tau, which causes a dose-dependent increase in tau aggregation (Van dermeeren et al., J Alzheimers Dis. (2018); 65(1): 265-281). Neutralization of tau inoculation by different anti-tau mAbs has been demonstrated in this model to be partially dependent on Tau's Fc-mediated ADP Figure 21A ) despite both anti-Tau mAbs neutralizing tau inoculation compared to isotype controls, a statistically significant difference was observed between the mAb with effector function (mouse IgG2a) and the mAb without effector function (mouse IgG2aσ (Vafa et al., Methods. 2014 Jan 1; 65(1): 114-26)), demonstrating the model's partial dependence on mAb effector function.

[0505] A similar study was performed comparing anti-Tau mAb PT1B844 with mouse IgG2a Fc to PT1B844 TTP mAb with human IgG1 AASFc. Co-injection of mAbs was utilized to standardize any differences in PK properties between the mAbs and TTP mAb. Both anti-Tau mAbs neutralized tau inoculation compared to isotype controls. The TTP mAb showed at least equivalent effect compared to the mAb with full Fc effector function, suggesting that non-canonical ADP mechanisms are functional in vivo Figure 21B ).

[0506] Stereotactic injection of PHF in P301L mice

[0507] PHF tau inoculation studies, including the presently described study, were performed in compliance with AAALAC guidelines, according to the local ethical committee (628-Tau Spread, Janssen Pharmaceutica) and nationally approved protocols. Mice expressing the longest human tau isoform with the P301L mutation (tau-4R / 2N-P301L) (Terwel et al., 2005; Peeraer et al., 2015) were individually housed in individually ventilated cages in a rich environment and under a 12 / 12 h light / dark cycle (light on at 6:00 AM). At 90 + / - 7 days of age, mice were randomized with respect to treatment group and gender and received a unilateral injection of AD-derived PHF in the right hippocampus (CA1) (anti-IgG2a (n = 19); anti-phospho Tau mouse IgG2a (n = 20) or anti-phospho Tau-TTE (n = 20).

[0508] Tau. P301L mice were deeply anesthetized with isoflurane (5% in 36% oxygen) and fixed in a stereotactic frame (Stoelting-Neurostar combination). During the further procedure, a 2% isoflurane level was maintained. Using a 30G syringe (Hamilton), 3 μL was injected at a speed of 0.25 μl / min at the selected coordinates in the right hemisphere: anteroposterior -2.0, mediolateral +1.6 from the bregma, dorsoventral 1.4 mm from the dura. Body weight was monitored before and after injection every week and no differences were observed between treatment and control groups for all injection experiments (not shown).

[0509] Two months after injection, mice were sacrificed by decapitation and brain tissue from the ipsilateral hemisphere was snap-frozen. Before extraction, tissue was weighed and homogenized in 600 μL buffer H / 100 mg tissue (10 mM Tris, 800 mM NaCl, 1 mM EGTA and 10% sucrose / pH 7.4). The homogenate was centrifuged at 27 000 x g for 20 min and the supernatant was frozen at -80°C.

[0510] Biochemical analysis MesoScale Discovery (MSD)

[0511] Coated antibodies (AT8) were diluted in PBS (1 pg / mL) and aliquoted into MSD plates (30 pL / well) (L15XA, MSD, Rockville, MD, USA), which were incubated overnight at 4°C. After washing with 5 x 200 pL of PBS / 0.5% Tween-20, the plates were blocked with 0.1% casein / PBS and washed again with 5 x 200 pL of PBS / 0.5% Tween-20. After addition of samples and standards (both diluted in 0.1% casein / PBS), the plates were incubated overnight at 4°C. Subsequently, the plates were washed with 5 x 200 pL of PBS / 0.5% Tween-20 and SULFO-TAG™ conjugated detection antibodies (AT8) in 0.1% casein / PBS were added and incubated for 2 h at room temperature while shaking at 600 rpm. After a final wash (5 x 200 pL of PBS / 0.5% Tween-20), 150 pL of 2x buffer T (MSD) were added and the plates were read out with the MSD imager. Raw signals were normalized to a standard curve consisting of 16 dilutions of a sarcosyl-insoluble preparation (PHF) from an autopsy AD brain and expressed as arbitrary units (AU) PHF. Statistical analysis was performed with GraphPad prism software (ANOVA followed by Bonferroni correction for multiple testing). P values < 0.05 were considered as significant differences.

[0512] Discussion

[0513] To achieve an optimized brain delivery platform based on receptor-mediated transcytosis, mAbs were generated that specifically bind to the human transferrin receptor (huTfR) in a pH-dependent manner with a certain range of affinities. The relationship between TfR binding affinity and transcytosis efficiency has been extensively covered in many publications, with the focus on the equilibrium dissociation constant K D dissociation constant K D is an important measure, the inventors have surprisingly demonstrated that the association kinetics k a and the dissociation rate k d are critical for transcytosis. The inventors found that for efficient transcytosis and pharmacodynamic activity of a delivered therapeutic mAb, both the association and dissociation rates need to be optimized. Based on this result, when, for example, k a ≥ 10 5 M −1 s −1 and the neutral k d = 2 x 10 -3 sec -1Optimal transcytosis occurs at this time. While not wishing to be bound by theory, it is hypothesized that the interplay between association and dissociation rates is critical to ensure efficient transcellular transport in polarized cells by a variety of intracellular vesicles responsible for protein transport.

[0514] It was demonstrated that administration of the tripod mAb in cynomolgus monkeys resulted in 6-12x increase in brain concentration compared to control mAbs. Increased acidic FcRn binding results in decreased peripheral clearance and increased brain concentration. Under normal physiological conditions, FcRn-mediated antibody export from the brain can be critical in maintaining brain homeostasis by avoiding unwanted inflammatory and immune responses in the brain (Schlachetzki, Zhu et al. 2002, Roopenian and Akilesh 2007). While the majority of evidence suggests a strong role for FcRn-mediated antibody export, some controversy remains regarding this clearance mechanism (Garg and Balthasar 2009, Abuqayyas and Balthasar 2013). The inventors found that increasing the binding affinity for FcRn has a positive impact on peripheral and brain concentration, suggesting that any enhanced export is negligible in this system. Dose response experiments using the tripod mAb in cynomolgus monkeys demonstrated the saturable capacity of the transport mechanism, which occurs at 30 mg / kg in this species. Dose response characterization with extensive repeat dosing was also performed in cynomolgus monkeys and will greatly aid in predicting human doses and the utility of this platform for specific therapeutic applications.

[0515] Reticulocyte depletion is a safety liability known for TfR binding antibodies. The inventors observed that with mAbs active for effector function, virtually acute and nearly complete reticulocyte depletion was observed. A number of approaches have been described to avoid this depletion, including reducing effector function {Couch, 2013 #589} and through molecular structure {Weber, 2018 #590}. Despite the inventors utilizing a structure very similar to one that has been described as spatially capable of attenuating peripheral effector function, they observed robust reticulocyte depletion with mAbs active for effector function.

[0516] A clear disadvantage of Fc mutagenesis is the elimination of effector function from therapeutic mAbs. For many therapeutic targets in the brain, such as beta-amyloid and Tau, ADP is thought to be important for efficacy. Previous studies have demonstrated that recycling receptors, including TfR, can be excluded from the sorting tubule and diverted to the lysosome by multivalent cargo binding (Marsh, 1995, J Cell Biol(1995) 129 (6): 1509-1522; Weflen, 2013 Mol Biol Cell. 2013 Aug 1; 24(15): 2398-24050. Inventors demonstrated that this endogenous transfer of multivalent cargo can serve as an alternative non-canonical non-FcyR mechanism of ADP. Tau internalized by non-canonical and canonical ADP is similarly trafficked in microglia, with Tau aggregates trafficked through the endolysosomal system to lysosomes for degradation. The non-canonical ADP can be used for various therapeutic applications where ADP is necessary for efficacy, but canonical ADP is detrimental for safety.

[0517] Data suggest that non-canonical ADP is more effective than canonical ADP, possibly due to inherent differences in binding and internalization between FcyR and TfR. FcyR-mediated internalization requires clustering of receptors by mAb, while TfR rapidly internalizes and recycles independent of mAb binding. A second possible explanation is macrophage and microglial depletion (Zent, 2017 FEBS J. 2017 Apr; 284(7):1021-1039). Macrophage depletion appears to depend on the length of time macrophages are exposed to the target (Church, Van Der Meid et al. 2016, Clin Exp Immunol. 2016 Jan; 183(1): 90-101) (Mukundan, 2009, Nat Med. 2009 Nov; 15(11): 1266-72), which is consistent with our observations (canonical ADP ceasing over time). Observations of macrophage depletion have been made in vitro and in patients, suggesting that this depletion phenotype can impact the therapeutic efficacy of mAbs with effector functions. Non-canonical ADP provides an efficacy benefit by mediating ADP without activating microglia through binding FcyR, avoiding this depletion phenotype.

[0518] Another benefit of non-canonical ADPs is that they occur without stimulating the production of pro-inflammatory cytokines by avoiding microglial activation. The safety of using effector-functionally active mAbs in the treatment of brain diseases remains controversial, particularly regarding the increased neuroinflammation in patients already suffering from chronic neuroinflammation (reviewed in {Heneka, 2015 #591}). Furthermore, there is increasing interest in the role of inflammation in the pathogenesis of neurodegenerative diseases involving increased inflammation, and its ability to participate in or further activate potentially exhausted microglia, which remains controversial. For example, the neurotoxic effects of canonical ADPs have been demonstrated, and it is hypothesized that effector-functionally active mAbs may pose safety risks {Lee, 2016 #592}. The non-canonical ADP mechanism described here avoids potential neuroinflammatory burden by enhancing the effective clearance of Tau without requiring microglial activation or stimulating the release of pro-inflammatory cytokines. In summary, robust brain delivery platforms have been characterized in terms of pharmacokinetics, pharmacodynamics, and safety, establishing robust preclinical characterization necessary to advance clinical trials.

[0519] When formatted and fused to the prototype anti-BACE (β-secretase) antagonist mAb as an scFv brain shuttle vector, compared to the anti-BACE mAb alone, in i.v. A 4-10x increase in brain concentration was observed after administration to transgenic mice expressing huTfR. A strong PK:PD relationship was also noted, with a dose-dependent decrease in β-amyloid protein detected. The optimal-performing brain shuttle vector enhanced brain delivery over competing molecules, achieving best-in-class delivery through optimized binding interactions between the brain shuttle vector and huTfR.

[0520] The optimized brain shuttle vector was then fused with PT1B844 (Tau-binding mAb). When in cynomolgus monkeys... i.v Upon administration, the brain shuttle fused with PT1B844 demonstrated a 6-16 fold increase in brain concentration. Similar to mouse data, this increase in brain concentration surpassed that of the best-performing brain shuttle reported in the literature. In addition to superior brain PK, the brain shuttle was modified to reduce Fc-mediated effector function and did not induce rapid reticulocyte depletion in cynomolgus monkeys, as has been reported by competitors. Importantly, the loss of Fc function did not affect the efficacy of therapeutic Tau mAbs, as the brain shuttle mediated microglial uptake of Tau more effectively than PT1B844 alone.

[0521] Those skilled in the art will understand that changes can be made to the above embodiments without departing from its broad inventive concept. Therefore, it should be understood that the invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined in the appended claims.

[0522] Citation

[0523] Abuqayyas, L. and J. P. Balthasar (2013). "Investigation of the role of FcgammaR and FcRn in mAb distribution to the brain." Mol Pharm 10(5): 1505-1513.

[0524] Church, A. K., K. R. VanDerMeid, N. A. Baig, A. M. Baran, T. E. Witzig, G. S. Nowakowski and C. S. Zent (2016). "Anti-CD20 monoclonal antibody-dependent phagocytosis of chronic lymphocytic leukaemia cells by autologous macrophages." Clin Exp Immunol 183(1): 90-101.

[0525] Cooper, P. R., G. J. Ciambrone, C. M. Kliwinski, E. Maze, L. Johnson, Q. Li, Y. Feng and P. J. Hornby (2013). "Efflux of monoclonal antibodies from rat brain by neonatal Fc receptor, FcRn." Brain Res 1534: 13-21.

[0526] Dall'Acqua, W. F., P. A. Kiener and H. Wu (2006). "Properties of human IgG1s engineered for enhanced binding to the neonatal Fc receptor (FcRn)." J Biol Chem 281(33): 23514-23524.

[0527] Garg, A. and J. P. Balthasar (2009). "Investigation of the influence of FcRn on the distribution of IgG to the brain." AAPS J 11(3): 553-557.

[0528] Goulatis, L. I. and E. V. Shusta (2017). "Protein engineering approaches for regulating blood-brain barrier transcytosis." Curr Opin Struct Biol 45:109-115.

[0529] Johnsen, K. B., A. Burkhart, F. Melander, P. J. Kempen, J. B. Vejlebo, P. Siupka, M. S. Nielsen, T. L. Andresen and T. Moos (2017). "Targeting transferrin receptors at the blood-brain barrier improves the uptake of immunoliposomes and subsequent cargo transport into the brain parenchyma." Sci Rep 7(1): 10396.

[0530] Robbie, G. J., R. Criste, W. F. Dall'acqua, K. Jensen, N. K. Patel, G. A. Losonsky and M. P. Griffin (2013). "A novel investigational Fc-modified humanized monoclonal antibody, motavizumab-YTE, has an extended half-life in healthy adults." Antimicrob Agents Chemother 57(12): 6147-6153.

[0531] Roopenian, D. C. and S. Akilesh (2007). "FcRn: the neonatal Fc receptor comes of age." Nat Rev Immunol 7(9): 715-725.

[0532] Schlachetzki, F., C. Zhu and W. M. Pardridge (2002). "Expression of the neonatal Fc receptor (FcRn) at the blood-brain barrier." J Neurochem 81(1):203-206.

[0533] Vandermeeren, M., M. Borgers, K. Van Kolen, C. Theunis, B. Vasconcelos, A. Bottelbergs, C. Wintmolders, G. Daneels, R. Willems, K. Dockx, L. Delbroek, A. Marreiro, L. Ver Donck, C. Sousa, R. Nanjunda, E. Lacy, T. Van De Casteele, D. Van Dam, P. P. De Deyn, J. A. Kemp, T. J. Malia and M. H. Mercken (2018). "Anti-Tau Monoclonal Antibodies Derived from Soluble and Filamentous Tau Show Diverse Functional Properties in vitro and in vivo." J Alzheimers Dis 65(1): 265-281.

[0534] Yu, Y. J., J. K. Atwal, Y. Zhang, R. K. Tong, K. R. Wildsmith, C. Tan, N. Bien-Ly, M. Hersom, J. A. Maloney, W. J. Meilandt, D. Bumbaca, K. Gadkar, K. Hoyte, W. Luk, Y. Lu, J. A. Ernst, K. Scearce-Levie, J. A. Couch, M. S. Dennis, and R. J. Watts (2014). "Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates." Sci Transl Med 6(261): 261ra154.

[0535] Yu, Y. J., Y. Zhang, M. Kenrick, K. Hoyte, W. Luk, Y. Lu, J. Atwal, J. M. Elliott, S. Prabhu, R. J. Watts, and M. S. Dennis (2011). "Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target." Sci Transl Med 3(84): 84ra44.

[0536] Zent, C. S., and M. R. Elliott (2017). "Maxed out macs: physiologic cell clearance as a function of macrophage phagocytic capacity." FEBS J 284(7): 1021-1039.

Claims

1. An anti-transferrin receptor (TfR) antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 292, 293, and 294, respectively, and a light chain variable region comprising light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 295, 296, and 297, respectively.

2. The anti-TfR antibody or antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment thereof is a single chain variable fragment (scFv) comprising a heavy chain variable region covalently linked to a light chain variable region by a linker.

3. The anti-TfR antibody or antigen-binding fragment thereof of claim 2, wherein the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

291.

4. The anti-TfR antibody or antigen-binding fragment thereof of claim 3, wherein the scFv comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

291.

5. A conjugate comprising the anti-TfR antibody or antigen-binding fragment thereof of any one of claims 1-4 conjugated to a therapeutic agent.

6. An isolated nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1-4 or the conjugate of claim 5.

7. A vector comprising the isolated nucleic acid of claim 6.

8. A host cell comprising the nucleic acid of claim 6 or the vector of claim 7.

9. A method of producing the antibody or antigen-binding fragment of any one of claims 1-4 or the conjugate of claim 5, comprising culturing a cell comprising a nucleic acid encoding the antibody or antigen-binding fragment or the conjugate under conditions wherein the antibody or antigen-binding fragment or the conjugate is produced, and recovering the antibody or antigen-binding fragment or the conjugate from the cell or cell culture.

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-4 or the conjugate of claim 5 and a pharmaceutically acceptable carrier.

11. An anti-TfR scFv, comprising: a heavy chain variable region comprising HCDRs HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 292, 293, and 294, respectively, and a light chain variable region comprising LCDRs LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 295, 296, and 297, respectively, wherein the scFv comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 291.

Images