USES OF AN ANTIBODY, METHOD FOR MAKING AN ANTIBODY AND ANTIBODIES

专利摘要:
methods to improve the safety of transport of the blood-brain barrier. the present invention relates to compositions and methods for enhancing the blood-brain barrier receptor-mediated transport safety of the blood-brain barrier.
公开号:BR112014028838A2
申请号:R112014028838
申请日:2013-05-20
公开日:2020-05-12
发明作者:Couch Jessica；Yu Zuchero Joy；Dennis Mark；Jefferson Watts Ryan
申请人:Genentech Inc；
IPC主号:

专利说明:

“USES OF AN ANTIBODY, METHOD FOR MAKING AN ANTIBODY AND ANTIBODIES”
Field of the Invention [001] The present invention relates to compositions and methods for improving the safety of blood-brain barrier receptor-mediated transport of the blood-brain barrier.
Background of the Invention [002] The penetration of drugs with large molecules into the brain is severely limited by the widely impermeable blood-brain barrier (BBB). Among the several strategies to overcome this obstacle is the use of the transitos trafficking pathways of endogenous receptors expressed in the capillary endothelium of the brain. Recombinant proteins such as monoclonal antibodies were designed against these receptors to allow receptor-mediated delivery of large molecules to the brain. Strategies to maximize absorption in the brain, while minimizing reverse transcytosis back into the blood, and also to maximize the degree of accumulation after therapeutic dosing, have been addressed with the discovery that antibodies with low affinity for BBB receptors offer the potential for substantially increase BBB transport and CNS retention of associated therapeutic components / molecules in relation to typical high-affinity antibodies for these receptors (Atwal et al., Sei. Transi. Med. 3, 84ra43 (2011); Yu et al. , Sei. Transi Med. May 25, 2011: Vol. 3, Edition 84, p. 84ra44). However, the safety of administering these antibodies and conjugates has not been fully explored.
Brief Description of the Invention [003] Monoclonal antibodies have great therapeutic potential for treating neurological or central nervous system (CNS) diseases,
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2/171 but their passage to the brain is limited by the blood-brain barrier (BBB). Previous studies have shown that a very small percentage (approximately 0.1%) of an IgG circulating in the bloodstream crosses the BBB in the CNS (Felgenhauer, Klin. Wschr. 52: 1158-1164 (1974)), where the concentration of the antibody in the CNS it may be insufficient to allow a strong effect. It was previously discovered that the percentage of the antibody it distributes in the CNS could be improved by scanning the BBB receptors (i.e., transferrin receptor, insulin receptor, protein 8 related to the low density lipoprotein receptor, glucose transporter 1 ( Glutl) and the like) (see, for example, WO 9502421). For example, the BBB anti-receptor antibody can be produced as multispecific to target one or more desired antigens on the CNS, or one or more heterologous molecules can be coupled to the BBB anti-receptor antibody; in both cases, the anti-BBB receptor antibody can assist in the distribution of a therapeutic molecule in the CNS through the BBB.
[004] However, targeting a BBB receptor with a traditional specific high affinity antibody has generally resulted in a limited increase in BBB transport. Later, it was discovered by the applicants that the magnitude of antibody absorption and the distribution in the CNS is inversely related to its binding affinity for the BBB receptor among the anti-BBB antibodies studied. For example, a low affinity antibody to the transferrin receptor (TfR) dosed at therapeutic dose levels greatly improves BBB transport and CNS retention of the anti-TfR antibody over a higher affinity anti-TfR antibody, and makes it is possible to more easily achieve therapeutic concentrations in the CNS (Atwal et al., Sei. Transi. Med. 3, 84ra43 (2011)). The proof of this transport in BBB was obtained with the use of a bispecific antibody that binds both to TfR
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3/171 as well as the amyloid precursor protein (APP) dividing enzyme, βsecretase (BACE1). A single systemic dose of the bispecific anti-TfR / BACE1 antibody genetically engineered using the methodology of the invention not only resulted in significant antibody absorption in the brain, but also dramatically reduced Αβ-Μο levels compared to bispecific antiBACE1 alone, suggesting that BBB penetration affects the potency of anti-BACE1. (Atwal et al., Sei. Transi. Med. 3, 84ra43 (2011); Yu et al., Sei. Transi. Med. 3, 84ra44 (2011)).
[005] These data and experiments highlighted several causative mechanisms behind the increased absorption of an antibody in the CNS using a less affinity antibody approach. First, high affinity BBB-receptor antibodies (BBB-R) (eg, anti-TfR ^a ) limit brain absorption by rapidly saturating BBB-R in the cerebral vasculature, thereby reducing the total amount of antibody absorbed in the brain and also restricting its distribution in the vasculature. Visibly, decreasing the affinity for BBB-R improves absorption and distribution in the brain, with a strong change observed in the location of the vasculature for neurons and associated neuropiles distributed in the CNS. Second, it is proposed that the lower affinity of the antibody to the BBB-R impairs the ability of the antibody to return to the vascular side of the BBB through the BBB-R from the side of the CNS membrane, due to the total affinity of the antibody to the BBB -R is low and the local antibody concentration on the BBB side of the CNS does not saturate due to the rapid dispersion of the antibody in the CNS compartment. Third, in vivo, and as seen for the TfR system, antibodies with less affinity for BBB-R are not cleared from the system efficiently, such as those with greater affinity for BBB-R, and thus maintain circulating concentrations greater than its most affinity counterpart. This is advantageous because the levels of circulating antibodies in the
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4/171 antibodies of lesser affinity are sustained at therapeutic levels for a longer period of time than the antibody of greater affinity, which consequently improves the absorption of the antibody in the brain for a longer period of time. In addition, this improvement in both plasma and brain exposure can reduce the frequency of dosing at the clinic, which would have a potential benefit not only for patient adherence to treatment and convenience, but also to mitigate any side effects or effects outside the target of the antibody and / or a therapeutic compound coupled thereto.
[006] The low-affinity BBB-R antibodies described in the work referenced above were selected / genetically engineered to avoid interference with the natural link between transferrin and TfR, and thus avoid potential side effects related to iron transport. However, after administration of these certain antibodies in mice, some strong side effects have been observed. The mice exhibited a primary response to a strong depletion of the reticulocyte population accompanied by the rapid onset of acute clinical symptoms, as described in the Examples. In addition, in vitro studies using a human erythroblast cell line and primary bone marrow cells treated with anti-TfR antibodies have shown that strong depletion of TfR-positive erythroid cells is also observed in human cell systems (see, for example, example, Example 7). Although mice recovered from both acute clinical symptoms and decreased reticulocyte levels in due course, avoiding or otherwise mitigating this impact on reticulocytes is clearly desirable for an anti-TfR antibody to be able to be used safely as a molecule. therapy.
[007] Consequently, the invention provides compositions and
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5/171 methods that reduce or eliminate undesirable reductions in the reticulocyte population by administering anti-TfR, while still allowing for improved transport in the BBB, increased distribution in the CNS and retention in the CNS provided by low-affinity anti-TfR antibodies administered in concentrations therapeutic. The results described in this application show that the main response to the administration of anti-TfR (strong depletion of reticulocytes and acute clinical signs) is driven largely by the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the antibody, while the Residual reticulocyte depletion effect is mediated by the complement pathway. Several general approaches to mitigate the observed effect of anti-TfR antibodies on both primary and residual reticulocyte depletion are provided in the present application, and can be used individually or in combination.
[008] In one approach, the effector function of the antiBBB-R antibody is reduced or eliminated in order to reduce or eliminate ADCC activity. In another approach, the affinity of the anti-BBB-R antibody to BBBR is further reduced, so that the interactions of the antibody with the reticulocyte population are less harmful to that population. A third approach is aimed at reducing the amount of antiBBB-R antibody that is present in the plasma, to reduce the exposure of the reticulocyte population to concentrations potentially harmful to the antibody. A fourth approach seeks to protect, stabilize and / or reconstitute the reticulocyte population, so that any potential depletion of the reticulocyte population by the administration of the anti-BBB-R antibody is avoided, reduced or mitigated.
[009] The reduction or elimination of the effector function, as described in the present application, can be accomplished through: (I) reduction or elimination of glycosylation of wild type mammalian antibody, (by
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6/171 example, by producing the antibody in an environment where such glycosylation cannot occur, by mutating one or more carbohydrate attachment points, so that the antibody cannot be glycosylated, or by chemical or enzymatic removal of one or more more carbohydrates from the antibody after it has been glycosylated); (ii) by reduction or elimination of the ability to bind to the Fc receptor (for example, by mutation of the Fc region, by deletion within the Fc region or deletion of the Fc region); or (iii) by using an antibody isotype known to have little or no effector function (i.e., including, but not limited to, IgG4).
[0010] The decrease in complement activation of the antibody, as described in this application, can be done by reducing or eliminating the C1q binding capacity of the anti-BBB-R antibody (for example, by mutation, deletion or elimination of the region Fc, or by modifying the non-Fc portion of the anti-BBB-R antibody), or otherwise by suppressing activation or activity of the complement system (for example, by co-administering one or more activation of the complement pathway or inhibitors of the complement pathway activity).
[0011] When the binding of the anti-BBB-R antibody to the BBB-R in reticulocytes or other cell types triggers its depletion, as the anti-TfR antibodies exemplified in the present application, the reduction of the binding of antibodies to BBB-R in reticulocytes or other cell types should, in turn, decrease the amount of depletion of reticulocytes or other cell types seen after administration of the antibody. In fact, this was demonstrated in the present application (see, for example, Figure 6B). The affinity of the anti-BBB-R antibody to BBB-R can be modified using any of the methods described in the present application and as shown in the Examples.
[0012] Reducing the amount of anti-BBB-R antibody present
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7/171 in plasma, in order to reduce the exposure of the reticulocyte population to potentially harmful concentrations of the antibody, can be performed in several ways. One method is to simply decrease the amount of antibody that is dosed, while potentially also increasing the frequency of dosing, so that the maximum plasma concentration is decreased, but a sufficient serum level is maintained for efficacy, although still below the effect threshold. collateral cell depletion. Another method, which can be combined with dosage modifications, is to select or genetically engineer an anti-TfR antibody that has a pH sensitive link to TfR, so that it binds to the TfR cell surface in plasma at pH 7.4, desirably with low affinity as described in the present application, but upon internalization in an endosomal compartment, this binding to TfR is rapidly and significantly reduced at a relatively lower pH of that compartment (pH 5.5 to 6.0). This dissociation can protect the antibody from antigen-mediated clearance, or increase the amount of antibody that is distributed to the CNS or recycled via the BBB - in both cases, the effective concentration of the antibody is increased over an anti-TfR antibody. that does not understand this pH sensitivity, without increasing the administered dose of the antibody.
[0013] Protecting, stabilizing and / or recomposing reticulocyte populations can be accomplished with the use of medications or physical methods. In addition to the anti-BBB-R antibody, at least one additional therapeutic agent can be co-administered (simultaneously or sequentially) that mitigates negative side effects of the antibody in the reticulocyte populations. Examples of such therapeutic agents include, but are not limited to, erythropoietin (EPO), iron supplements, vitamin C, folic acid and vitamin B12. The physical recomposition of red blood cells (ie, reticulocytes) is also possible, for example, by transfusion of similar cells, which
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8/171 may be from another individual of similar blood type or may have been extracted from the subject to which the anti-BBB-R antibody is administered.
[0014] A person skilled in the art will appreciate that any combination of the above methods can be employed to genetically engineer an antibody (and / or dosage regimen for it) with an optimal balance between (!) The low desirable affinity for BBB- R which will maximize the transport of the antibody and any conjugated compounds to the CNS; (ii) the affinity of the conjugated compounds (including, as a non-limiting example, a second or additional antigen binding specificity in the anti-TfR antibody) for your CNS antigen, as this is relevant to the amount of the compound that needs to be present on the CNS to have a therapeutic effect; (iii) the clearance rate of the anti-BBB-R antibody; and (iv) the impact on reticulocyte populations.
[0015] It will also be appreciated that the recognized effect of depletion of reticulocytes in the present application for administration of anti-TfR antibody may be useful in the treatment of any disease or dysfunction where reticulocyte overproliferation is problematic. For example, in congenital polycythemia or neoplastic polycythemia vera, the increase in red blood cell count due to hyperproliferation of, for example, reticulocytes, results in blood thickening and the accompanying physiological symptoms. Administration of an anti-TfR antibody of the invention in which at least the partial effector function of the antibody has been preserved would allow selective removal of immature reticulocytes without affecting normal transferrin transport in the CNS. The dosage of this antibody could be modulated, so that these acute clinical symptoms could be minimized (that is, by dosing a very low dose or at widely spaced intervals), as is well understood in the art.
[0016] Each of the anti-TfR / BACE1 and anti-TfR / Abeta is a
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9/171 new and promising therapeutic candidate for the treatment of Alzheimer's disease. In addition, bispecific targeting technology based on receptor-mediated transport (RMT) opens the door to a wide variety of therapeutic possibilities for CNS diseases. The invention provides genetically engineered therapeutic methods of BBB penetration that greatly improve the transport through the BBB and distribution in the CNS of the therapeutic without reticulocyte depletion.
[0017] Consequently, in a first embodiment, the invention provides a method of transporting a compound across the blood-brain barrier in a subject, which comprises exposing an antibody that binds with low affinity to a blood-brain barrier receptor (BBB-R) coupled to a compound for the blood-brain barrier, so that the antibody transports the compound coupled to it through the blood-brain barrier, in which the reduction of red blood cell levels in the subject by administering the antibody to the subject is decreased or eliminated. In one aspect, BBB-R is selected from the group consisting of transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF receptor), protein 8 related to the low lipoprotein receptor density (LRP8), protein 1 related to the low density lipoprotein receptor (LRP1), glucose transporter 1 (Glutl) and growth factor similar to heparin-binding epidermal growth factor (HB-EGF). In another aspect, BBB-R is a human BBB-R. In one aspect, BBB-R is TfR. In another aspect, BBB-R is TfR, and the antibody does not inhibit TfR activity. In another aspect, BBB-R is TfR, and the antibody does not inhibit TfR activity to transferrin.
[0018] In another aspect, red blood cells are immature red blood cells. In another aspect, immature red blood cells are reticulocytes. In another aspect, the reduction in reticulocyte levels is
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10/171 accompanied by acute clinical symptoms. In another aspect, the method also comprises the stage of monitoring the subject by red blood cell depletion.
[0019] In another aspect, one or more properties of the antibody have been modified to reduce the impact of the antibody on reticulocyte levels and / or to reduce the severity or presence of acute clinical symptoms in the subject. In one aspect, the antibody's affinity for BBB-R is modified, that is, decreased. In another aspect, the effector function of the antibody's Fc region is modified. In one aspect, the effector function has been reduced or eliminated in relation to the effector function of a wild type antibody of the same isotype. In another aspect, the effector function is reduced or eliminated by reducing glycosylation of the antibody. In another aspect, the glycosylation of the antibody is reduced by production of the antibody in an environment that does not allow for wild-type glycosylation. In one aspect, the antibody is produced in a non-mammalian cell production system. In another aspect, the antibody is produced synthetically. In another aspect, the glycosylation of the antibody is reduced by removing carbohydrate groups already present in the antibody. In another aspect, the glycosylation of the antibody is reduced by modifying the antibody so that wild-type glycosylation does not occur. In another aspect, the antibody's Fc region comprises a mutation at position 297, so that the wild-type asparagine residue at that position is replaced by another amino acid that interferes with glycosylation at that position. In another aspect, the effector function is reduced or eliminated by modifying the antibody isotype to an isotype that naturally has reduced or eliminated effector function.
[0020] In another aspect, the Fc region is modified to reduce or eliminate the effector function. In one aspect, the effector function is reduced or eliminated by at least one modification of the Fc region. In one respect,
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11/171 modification is a point mutation of the Fc region to impair binding to one or more Fc receptors selected from the following positions: 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272 , 278, 289, 292, 293, 294, 295, 296, 297, 298, 301,303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416 , 419, 434, 435, 437, 438 and 439. In another aspect, the modification is elimination of some or all of the Fc regions. In another aspect, the effector function is reduced or eliminated by deletion of all or a portion of the Fc region, or by genetic elaboration of the antibody, so that it does not include a competent Fc region for effector function. In one aspect, the antibody is selected from a Fab or a single chain antibody.
[0021] In one aspect, the antibody's Fc and / or non-Fc region is modified to reduce or eliminate activation of the complement pathway by the antibody. In one aspect, the modification is a point mutation in the Fc region to impair binding to C1q, selected from the following positions: 270, 322, 329 and 321. In another aspect, the modification is elimination of some or all of the Fc regions. In another aspect, the complement activation function is reduced or eliminated by deletion of all or a portion of the Fc region, or by genetic elaboration of the antibody, so that it does not include an Fc region that engages in the complement pathway. In one aspect, the antibody is selected from a Fab or a single chain antibody. In another aspect, the non-Fc region of the antibody is modified to reduce or eliminate activation of the complement pathway by the antibody. In one aspect, the modification is a point mutation of the CH1 region to impair binding to C3. In one aspect, the point mutation is at position 132 (see, for example, Vidarte et al., (2001) J. Biol. Chem. 276 (41): 3821738223).
[0022] In another aspect, the amount of dose and / or frequency of
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12/171 administration of the antibody is modulated to reduce the concentration of the antibody to which red blood cells are exposed. In another aspect, the antibody is modified to comprise pH-sensitive BBB-R binding.
[0023] In another aspect, an additional compound is administered in addition to the antibody and the coupled compound. In one aspect, the additional compound is responsible for or contributes to the failure to reduce reticulocyte levels. In another aspect, the additional compound inhibits or prevents activation or activity of the complement pathway (see, for example, Mollnes and Kirschfink (2006) Molec. Immunol. 43: 107-121). In another aspect, the compound also protects reticulocytes from antibody-related depletion. In another aspect, the additional compound supports the growth, development or restoration of reticulocytes. In another aspect, the additional compound is selected from erythropoietin (EPO), an iron supplement, vitamin C, folic acid and vitamin B12. In another aspect, the additional compound is red blood cells or reticulocytes from the same subject. In another aspect, the additional compound is another subject's red blood cells or reticulocytes.
[0024] In another aspect, the compound is a neurological dysfunction drug. In another aspect, the compound is an imaging agent. In another aspect, the compound is labeled. In another aspect, the antibody is labeled. In another aspect, the antibody does not impair the binding of BBB-R to one or more of its native ligands. In another aspect, the antibody binds specifically to TfR, so that it does not inhibit the binding of TfR to transferrin. In another aspect, the BBB is in a mammal. In another aspect, the mammal is a human. In another aspect, the mammal has neurological dysfunction. In another aspect, neurological dysfunction 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 , syndrome
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13/171
Angelman, Liddle's syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer and traumatic brain injury. In another aspect, the BBB is in a human.
[0025] In another aspect, the antibody has an IC50 for BBB-R of about 1 nM to about 100 μΜ. In another aspect, the IC50 is about 5 nM to about 100 μΜ. In another aspect, the IC50 is about 50 nM to about 100 μΜ. In another aspect, the IC50 is about 100 nM to about 100 μΜ. In another aspect, the antibody has an BBB-R affinity of about 5 nM to about 50 μΜ. In another aspect, the antibody has an affinity for BBB-R from about 30 nM to about 30 μΜ. In another aspect, the antibody, when coupled to a compound, has an affinity for BBB-R from about 30 nM to about 1 μΜ. In another aspect, the antibody, when coupled to a compound, has an affinity for the BBB-R from about 50 nM to about 1 μΜ. In another aspect, the antibody coupled to the complex specifically binds TfR and has an affinity for TfR between the observed affinities for the anti-TfR ^A / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In another aspect, the antibody coupled to the complex binds specifically to TfR and has an affinity for TfR between the observed affinities for the anti-TfR ^D / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In another aspect, the antibody coupled to the complex specifically binds to TfR and has an IC50 for TfR among the IC50s observed for the anti-TfR ^A / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In another aspect, the antibody coupled to the complex binds specifically to TfR and has an IC50 for TfR among the IC50s observed for the anti-TfR ^D / BACE1 antibody and the antiTfR ^E / BACE1 antibody. In one aspect, the affinity of the anti-BBB-R or anti-BBBR / compound for the BBB-R is measured using Scatchard analysis. In another aspect, the affinity of anti-BBB-R or anti-BBB-R / compound for BBBR is measured using BIACORE analysis. In another aspect, the affinity of the
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14/171 anti-BBB-R or anti-BBB-R / compound for the BBB-R is measured with the use of a competition ELISA.
[0026] In another aspect, the dissociation half-life of the BBB-R antibody to which it specifically binds is about 30 seconds to about 30 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 20 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 10 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 5 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 3 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 2 minutes. In another aspect, the dissociation half-life is about 2 minutes. In another aspect, the dissociation half-life is 1 minute or less. In another aspect, the antibody coupled to the compound specifically binds to TfR and has a dissociation half-life for TfR between the dissociation half-lives observed for the anti-TfR ^A 7BACE1 antibody and the anti-TfR ^E / BACE1 antibody of their respective connections to TfR. In another aspect, the antibody coupled to the compound specifically binds to TfR and has a dissociation half-life for TfR between the dissociation half-lives observed for the anti-TfR ^D / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. their respective connections to TfR. In another aspect, the dissociation half-life of the anti-BBB-R or anti-BBB-R / compound for the BBB-R is measured using BIACORE analysis. In another aspect, the dissociation half-life of the anti-BBB-R or anti-BBB-R / compound for the BBBR is measured using a competition binding assay, such as a competition ELISA. In another aspect, the antibody coupled to the compound is administered in a therapeutic dose. In one aspect, the therapeutic dose is a dose that saturates the BBB-R to which the antibody specifically binds. In another aspect, the antibody coupled to the compound is administered in a dose
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15/171 and a dose frequency that minimizes the interaction of red blood cells with the antibody coupled to the compound although it still facilitates the distribution of the compound through the BBB in the CNS at therapeutic levels.
[0027] In another aspect, the compound is covalently coupled to the antibody. In one aspect, the compound is linked to the antibody by a linker. In one aspect, the binder is cleavable. In another aspect, the binder is not cleavable. In another aspect, the compound is bound directly to the antibody. In one aspect, the antibody is a multispecific antibody and the compound optionally forms a portion of the multispecific antibody. In another aspect, the multispecific antibody comprises a first antigen binding site that binds to BBB-R and a second antigen binding site that binds to a brain antigen. In another aspect, the cerebral antigen is selected from the group consisting of: beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER2), Tau , apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine repeat rich kinase 2 (LRRK2), parchin, presenilin 1, presenilin 2, gamma secretase, receptor 6 of death (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), interleukin receptor 6 (IL6R), TNF receptor 1 (TNFR1), beta 1 interleukin and caspase 6. In another aspect, the multispecific antibody binds both to TfR like BACE1. In another aspect, the multispecific antibody binds to both TfR and Abeta. In another aspect, the multispecific antibody is labeled. In another aspect, the compound is reversibly coupled to the antibody, so that the compound is released from the antibody with or after transport in the BBB.
[0028] It will be appreciated that any of the aspects mentioned above can be applied individually or in combination with the aforementioned achievement.
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[0029] In another embodiment, the invention provides a method for increasing exposure in a subject's CNS to a compound, in which the compound is coupled to an antibody that binds with low affinity to a BBB-R, through this by increasing the exposure in the CNS to the compound, and in which the reduction of red blood cell levels in the subject by administering the antibody coupled to the compound to the subject is decreased or eliminated. In one aspect, BBB-R is selected from the group consisting of transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF receptor), protein 8 related to the low lipoprotein receptor density (LRP8), protein 1 related to the low density lipoprotein receptor (LRP1), glucose transporter 1 (Glutl) and growth factor similar to heparin-binding epidermal growth factor (HB-EGF). In another aspect, BBB-R is a human BBB-R. In one aspect, BBB-R is TfR. In another aspect, BBB-R is TfR, and the antibody does not inhibit TfR activity. In another aspect, BBB-R is TfR, and the antibody does not inhibit TfR activity to transferrin.
[0030] In another aspect, red blood cells are immature red blood cells. In another aspect, immature red blood cells are reticulocytes. In another aspect, the reduction in reticulocyte levels is accompanied by acute clinical symptoms. In another aspect, the method also comprises the stage of monitoring the subject by red blood cell depletion.
[0031] In another aspect, one or more properties of the antibody have been modified to reduce the impact of the antibody on reticulocyte levels and / or to reduce the severity or presence of acute clinical symptoms in the subject. In one aspect, the antibody's affinity for BBB-R is modified, that is, decreased. In another aspect, the effector function of the antibody's Fc region is modified. In one aspect, the effector function was reduced or
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17/171 eliminated in relation to the effector function of a wild type antibody of the same isotype. In another aspect, the effector function is reduced or eliminated by reducing glycosylation of the antibody. In another aspect, the glycosylation of the antibody is reduced by production of the antibody in an environment that does not allow for wild-type glycosylation. In one aspect, the antibody is produced in a non-mammalian cell production system. In another aspect, the antibody is produced synthetically. In another aspect, the glycosylation of the antibody is reduced by removing carbohydrate groups already present in the antibody. In another aspect, the glycosylation of the antibody is reduced by modifying the antibody so that wild-type glycosylation does not occur. In another aspect, the antibody Fc region comprises a mutation at position 297, so that the wild-type asparagine residue at that position is replaced by another amino acid that interferes with glycosylation at that position. In another aspect, the effector function is reduced or eliminated by modifying the antibody isotype to an isotype that naturally has reduced or eliminated effector function.
[0032] In another aspect, the Fc region is modified to reduce or eliminate the effector function. In another aspect, the effector function is reduced or eliminated by at least one modification of the Fc region. In one aspect, the modification is a point mutation in the Fc region to impair binding to one or more Fc receptors selected from the following positions: 238, 239, 248, 249, 252, 254, 265, 268, 269, 270 , 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301,303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414 , 416, 419, 434, 435, 437, 438 and 439. In another aspect, the modification is elimination of some or all of the Fc regions. In another aspect, the effector function is reduced or eliminated by deletion of all or a portion of the Fc region, or by genetic elaboration of the antibody, so that it does not include a competent Fc region for effector function. On a
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18/171 aspect, the antibody is selected from a Fab or a single chain antibody.
[0033] In one aspect, the antibody's Fc and / or non-Fc region is modified to reduce or eliminate activation of the complement pathway by the antibody. In one aspect, the modification is a point mutation in the Fc region to impair binding to C1q, selected from the following positions: 270, 322, 329 and 321. In another aspect, the modification is elimination of some or all of the Fc regions. In another aspect, the complement activation function is reduced or eliminated by deletion of all or a portion of the Fc region, or by genetic elaboration of the antibody, so that it does not include an Fc region that engages in the complement pathway. In one aspect, the antibody is selected from a Fab or a single chain antibody. In another aspect, the non-Fc region of the antibody is modified to reduce or eliminate activation of the complement pathway by the antibody. In one aspect, the modification is a point mutation of the CH1 region to impair binding to C3. In one aspect, the point mutation is at position 132 (see, for example, Vidarte et al, (2001) J. BioL Chem. 276 (41): 3821738223).
[0034] In another aspect, the amount of dose and / or frequency of administration of the antibody is modulated to reduce the concentration of the antibody to which the red blood cells are exposed. In another aspect, the antibody is modified to comprise pH-sensitive BBB-R binding.
[0035] In another aspect, an additional compound is administered in addition to the antibody and the coupled compound. In one aspect, the additional compound is responsible for or contributes to the failure to reduce reticulocyte levels. In another aspect, the additional compound inhibits or prevents activation or activity of the complement pathway (see, for example, Mollnes and Kirschfink (2006) Molec. Immunol. 43: 107-121). In another aspect, the compound
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19/171 still protects reticulocytes from antibody-related depletion. In another aspect, the additional compound supports the growth, development or restoration of reticulocytes. In another aspect, the additional compound is selected from erythropoietin (EPO), an iron supplement, vitamin C, folic acid and vitamin B12. In another aspect, the additional compound is red blood cells or reticulocytes from the same subject. In another aspect, the additional compound is another subject's red blood cells or reticulocytes.
[0036] In another aspect, the compound is a neurological dysfunction drug. In another aspect, the compound is an imaging agent. In another aspect, the compound is labeled. In another aspect, the antibody is labeled. In another aspect, the antibody does not impair the binding of BBB-R to one or more of its native ligands. In another aspect, the antibody binds specifically to TfR, so that it does not inhibit the binding of TfR to transferrin. In another aspect, the compound coupled to the antibody is administered to a mammal. In another aspect, the mammal is a human. In another aspect, the mammal has neurological dysfunction. In another aspect, neurological dysfunction 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, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer and traumatic brain injury.
[0037] In another aspect, the increase in CNS exposure to the compound is measured in relation to the CNS exposure of a compound coupled to a typical antibody that has no decreased affinity for BBB-R. In another aspect, the increase in CNS exposure to the compound is measured as a ratio of the amount of the compound found in the CNS to the amount found in the serum after administration. In another aspect, the increase in CNS exposure results in a rate greater than 0.1%. In another
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As an aspect, the increase in CNS exposure to the compound is measured in relation to the CNS exposure of a compound in the absence of a coupled antibody. In another aspect, the increase in CNS exposure to the compound is measured by imaging. In another aspect, the increase in CNS exposure to the compound is measured by an indirect reading, such as a change in one or more physiological symptoms.
[0038] In another aspect, the antibody has an IC50 for BBB-R of about 1 nM to about 100 μΜ. In another aspect, the IC50 is about 5 nM to about 100 μΜ. In another aspect, the IC50 is about 50 nM to about 100 μΜ. In another aspect, the IC50 is about 100 nM to about 100 μΜ. In another aspect, the antibody has an BBB-R affinity of about 5 nM to about 50 μΜ. In another aspect, the antibody has an affinity for BBB-R from about 30 nM to about 30 μΜ. In another aspect, the antibody, when coupled to a compound, has an affinity for BBB-R from about 30 nM to about 1 μΜ. In another aspect, the antibody, when coupled to a compound, has an affinity for the BBB-R from about 50 nM to about 1 μΜ. In another aspect, the antibody coupled to the complex binds specifically to TfR and has an affinity for TfR between the observed affinities for the anti-TfR ^A 7BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In another aspect, the antibody coupled to the complex binds specifically to TfR and has an affinity for TfR between the observed affinities for the anti-TfR ^D / BACE1 antibody and the anti-TfR ^ BACEI antibody. In another aspect, the antibody coupled to the complex specifically binds to TfR and has an IC50 for TfR among the IC50s observed for the anti-TfR ^A / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In another aspect, the antibody bound to the complex specifically binds the TfR and has an IC50 for TfR observed between the IC50 for anti-TfR antibody ^D / BACE1 and antiTíR antibody ^and / BACE1. In one respect, the affinity of anti-BBB-R or anti-BBB
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R / compound for the BBB-R is measured using Scatchard analysis. In another aspect, the affinity of anti-BBB-R or anti-BBB-R / compound for BBBR is measured using BIACORE analysis. In another aspect, the affinity of the anti-BBB-R or anti-BBB-R / compound for the BBB-R is measured with the use of a competition ELISA.
[0039] In another aspect, the dissociation half-life of the BBB-R antibody to which it specifically binds is about 30 seconds to about 30 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 20 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 10 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 5 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 3 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 2 minutes. In another aspect, the dissociation half-life is about 2 minutes. In another aspect, the dissociation half-life is 1 minute or less. In another aspect, the antibody coupled to the compound specifically binds to TfR and has a dissociation half-life for TfR between the dissociation half-lives observed for the anti-TfR ^A / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. their respective connections to TfR. In another aspect, the antibody coupled to the compound specifically binds to TfR and has a dissociation half-life for TfR between the dissociation half-lives observed for the anti-TfR ^D / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. their respective connections to TfR. In another aspect, the dissociation half-life of the anti-BBB-R or anti-BBB-R / compound for the BBB-R is measured using BIACORE analysis. In another aspect, the dissociation half-life of the anti-BBB-R or anti-BBB-R / compound for the BBBR is measured using a competition binding assay, such as a competition ELISA.
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22/171 [0040] In another aspect, the antibody coupled to the compound is administered in a therapeutic dose. In one aspect, the therapeutic dose is a dose that saturates the BBB-R to which the antibody specifically binds. In another aspect, the antibody coupled to the compound is administered at a dose and dose frequency that minimizes the interaction of red blood cells with the antibody coupled to the compound although it still facilitates the distribution of the compound through the BBB in the CNS at therapeutic levels.
[0041] In another aspect, the compound is covalently coupled to the antibody. In one aspect, the compound is linked to the antibody by a linker. In one aspect, the binder is cleavable. In another aspect, the binder is not cleavable. In another aspect, the compound is bound directly to the antibody. In one aspect, the antibody is a multispecific antibody and the compound optionally forms a portion of the multispecific antibody. In another aspect, the multispecific antibody comprises a first antigen binding site that binds to BBB-R and a second antigen binding site that binds to a brain antigen. In another aspect, the cerebral antigen is selected from the group consisting of: beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER2), Tau , apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine repeat rich kinase 2 (LRRK2), parchin, presenilin 1, presenilin 2, gamma secretase, receptor 6 of death (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), interleukin receptor 6 (IL6R), TNF receptor 1 (TNFR1), beta 1 interleukin and caspase 6. In another aspect, the multispecific antibody binds both to TfR like BACE1. In another aspect, the multispecific antibody binds to both TfR and Abeta. In another aspect, the multispecific antibody is labeled. In another aspect, the compound is reversibly coupled to the antibody, so that the compound is
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23/171 released from the antibody together with or after transport in the BBB.
[0042] It will be appreciated that any of the aspects mentioned above can be applied individually or in combination with the aforementioned achievement.
[0043] In another embodiment, the invention provides a method for decreasing the release of a compound administered to a subject, in which the compound is coupled to an antibody that binds with low affinity to a BBB-R, so that the release of the compound is decreased, and in which the reduction of red blood cell levels in the subject by administering the antibody coupled to the compound to the subject is decreased or eliminated. In one aspect, BBB-R is selected from the group consisting of transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF receptor), protein 8 related to the low lipoprotein receptor density (LRP8), protein 1 related to the low density lipoprotein receptor (LRP1), glucose transporter 1 (Glutl) and growth factor similar to heparin-binding epidermal growth factor (HB-EGF). In another aspect, BBB-R is a human BBB-R. In one aspect, BBB-R is TfR. In another aspect, BBB-R is TfR, and the antibody does not inhibit TfR activity. In another aspect, BBB-R is TfR, and the antibody does not inhibit TfR activity to transferrin.
[0044] In another aspect, red blood cells are immature red blood cells. In another aspect, immature red blood cells are reticulocytes. In another aspect, the reduction in reticulocyte levels is accompanied by acute clinical symptoms. In another aspect, the method also comprises the stage of monitoring the subject by red blood cell depletion.
[0045] In another aspect, one or more properties of the antibody have been modified to reduce the impact of the antibody on levels of
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24/171 reticulocytes and / or reduce the severity or presence of acute clinical symptoms in the subject. In one aspect, the antibody's affinity for BBB-R is modified, that is, decreased. In another aspect, the effector function of the antibody's Fc region is modified. In one aspect, the effector function has been reduced or eliminated in relation to the effector function of a wild type antibody of the same isotype. In another aspect, the effector function is reduced or eliminated by reducing glycosylation of the antibody. In another aspect, the glycosylation of the antibody is reduced by production of the antibody in an environment that does not allow for wild-type glycosylation. In one aspect, the antibody is produced in a non-mammalian cell production system. In another aspect, the antibody is produced synthetically. In another aspect, the glycosylation of the antibody is reduced by removing carbohydrate groups already present in the antibody. In another aspect, the glycosylation of the antibody is reduced by modifying the antibody so that wild-type glycosylation does not occur. In another aspect, the antibody Fc region comprises a mutation at position 297, so that the wild-type asparagine residue at that position is replaced by another amino acid that interferes with glycosylation at that position. In another aspect, the effector function is reduced or eliminated by modifying the antibody isotype to an isotype that naturally has reduced or eliminated effector function.
[0046] In another aspect, the Fc region is modified to reduce or eliminate the effector function. In another aspect, the effector function is reduced or eliminated by at least one modification of the Fc region. In one aspect, the modification is a point mutation in the Fc region to impair binding to one or more Fc receptors selected from the following positions: 238, 239, 248, 249, 252, 254, 265, 268, 269, 270 , 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301,303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414 , 416, 419, 434, 435, 437, 438 and 439. In another aspect, the
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25/171 modification is elimination of some or all of the Fc regions. In another aspect, the effector function is reduced or eliminated by deletion of all or a portion of the Fc region, or by genetic elaboration of the antibody, so that it does not include a competent Fc region for effector function. In one aspect, the antibody is selected from a Fab or a single chain antibody.
[0047] In one aspect, the antibody's Fc and / or non-Fc region is modified to reduce or eliminate activation of the complement pathway by the antibody. In one aspect, the modification is a point mutation in the Fc region to impair binding to C1q, selected from the following positions: 270, 322, 329 and 321. In another aspect, the modification is elimination of some or all of the Fc regions. In another aspect, the complement activation function is reduced or eliminated by deletion of all or a portion of the Fc region, or by genetic elaboration of the antibody, so that it does not include an Fc region that engages in the complement pathway. In one aspect, the antibody is selected from a Fab or a single chain antibody. In another aspect, the non-Fc region of the antibody is modified to reduce or eliminate activation of the complement pathway by the antibody. In one aspect, the modification is a point mutation of the CH1 region to impair binding to C3. In one aspect, the point mutation is at position 132 (see, for example, Vidarte et al, (2001) J. BioL Chem. 276 (41): 3821738223).
[0048] In another aspect, the amount of dose and / or frequency of administration of the antibody is modulated to reduce the concentration of the antibody to which the red blood cells are exposed. In another aspect, the antibody is modified to comprise pH-sensitive BBB-R binding.
[0049] In another aspect, an additional compound is administered in addition to the antibody and the coupled compound. In one respect, the compound
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26/171 is responsible for or contributes to the lack of reduction in reticulocyte levels. In another aspect, the additional compound inhibits or prevents activation or activity of the complement pathway (see, for example, Mollnes and Kirschfink (2006) Molec. Immunol. 43: 107-121). In another aspect, the compound also protects reticulocytes from antibody-related depletion. In another aspect, the additional compound supports the growth, development or restoration of reticulocytes. In another aspect, the additional compound is selected from erythropoietin (EPO), an iron supplement, vitamin C, folic acid and vitamin B12. In another aspect, the additional compound is red blood cells or reticulocytes from the same subject. In another aspect, the additional compound is another subject's red blood cells or reticulocytes.
[0050] In another aspect, the compound is a neurological dysfunction drug. In another aspect, the compound is an imaging agent. In another aspect, the compound is labeled. In another aspect, the antibody is labeled. In another aspect, the antibody does not impair the binding of BBB-R to one or more of its native ligands. In another aspect, the antibody binds specifically to TfR, so that it does not inhibit the binding of TfR to transferrin. In another aspect, the subject is a mammal. In another aspect, the mammal is a human. In another aspect, the mammal has neurological dysfunction. In another aspect, neurological dysfunction 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, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer and traumatic brain injury.
[0051] In another aspect, the decrease in the clearance of the compound is measured in relation to the clearance of a compound coupled to a typical antibody that has no decreased affinity for BBB-R. In another aspect, the
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Decrease in compound clearance is measured relative to compound clearance in the absence of a coupled antibody.
[0052] In another aspect, the antibody has an IC 50 for BBB-R of about 1 nM to about 100 μΜ. In another aspect, the IC50 is about 5 nM to about 100 μΜ. In another aspect, the IC50 is about 50 nM to about 100 μΜ. In another aspect, the IC50 is about 100 nM to about 100 μΜ. In another aspect, the antibody has an BBB-R affinity of about 5 nM to about 50 μΜ. In another aspect, the antibody has an affinity for BBB-R from about 30 nM to about 30 μΜ. In another aspect, the antibody, when coupled to a compound, has an affinity for BBB-R from about 30 nM to about 1 μΜ. In another aspect, the antibody, when coupled to a compound, has an affinity for the BBB-R from about 50 nM to about 1 μΜ. In another aspect, the antibody coupled to the complex binds specifically to TfR and has an affinity for TfR between the observed affinities for the anti-TfR ^A 7BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In another aspect, the antibody coupled to the complex binds specifically to TfR and has an affinity for TfR between the observed affinities for the anti-TfR ^D / BACE1 antibody and the anti-TfR ^ BACEI antibody. In another aspect, the antibody coupled to the complex specifically binds to TfR and has an IC50 for TfR among the IC50s observed for the anti-TfR ^A / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In another aspect, the antibody bound to the complex specifically binds the TfR and has an IC50 for TfR observed between the IC50 for anti-TfR antibody ^D / BACE1 and antiTíR antibody ^and / BACE1. In one aspect, the affinity of the anti-BBB-R or anti-BBBR / compound for the BBB-R is measured using Scatchard analysis. In another aspect, the affinity of anti-BBB-R or anti-BBB-R / compound for BBBR is measured using BIACORE analysis. In another aspect, the affinity of anti-BBB-R or anti-BBB-R / compound for BBB-R is measured with the use of
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Competition ELISA.
[0053] In another aspect, the dissociation half-life of the BBB-R antibody to which it specifically binds is about 30 seconds to about 30 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 20 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 10 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 5 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 3 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 2 minutes. In another aspect, the dissociation half-life is about 2 minutes. In another aspect, the dissociation half-life is 1 minute or less. In another aspect, the antibody coupled to the compound specifically binds to TfR and has a dissociation half-life for TfR between the dissociation half-lives observed for the anti-TfR ^A / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. their respective connections to TfR. In another aspect, the antibody coupled to the compound specifically binds to TfR and has a dissociation half-life for TfR between the dissociation half-lives observed for the anti-TfR ^D / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. their respective connections to TfR. In another aspect, the dissociation half-life of the anti-BBB-R or anti-BBB-R / compound for the BBB-R is measured using BIACORE analysis. In another aspect, the dissociation half-life of the anti-BBB-R or anti-BBB-R / compound for the BBBR is measured using a competition binding assay, such as a competition ELISA.
[0054] In another aspect, the antibody coupled to the compound is administered in a therapeutic dose. In one aspect, the therapeutic dose is a dose that saturates the BBB-R to which the antibody specifically binds. In another aspect, the antibody coupled to the compound is administered in a dose
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29/171 and a dose frequency that minimizes the interaction of red blood cells with the antibody coupled to the compound although it still facilitates the distribution of the compound through the BBB in the CNS at therapeutic levels.
[0055] In another aspect, the compound is covalently coupled to the antibody. In one aspect, the compound is linked to the antibody by a linker. In one aspect, the binder is cleavable. In another aspect, the binder is not cleavable. In another aspect, the compound is bound directly to the antibody. In one aspect, the antibody is a multispecific antibody and the compound optionally forms a portion of the multispecific antibody. In another aspect, the multispecific antibody comprises a first antigen binding site that binds to BBB-R and a second antigen binding site that binds to a brain antigen. In another aspect, the brain antigen is selected from the group consisting of: beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER2), Tau , apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine repeat rich kinase 2 (LRRK2), parchin, presenilin 1, presenilin 2, gamma secretase, receptor 6 of death (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), interleukin receptor 6 (IL6R), TNF receptor 1 (TNFR1), beta 1 interleukin and caspase 6. In another aspect, the multispecific antibody binds both to TfR like BACE1. In another aspect, the multispecific antibody binds to both TfR and Abeta. In another aspect, the multispecific antibody is labeled. In another aspect, the compound is reversibly coupled to the antibody, so that the compound is released from the antibody with or after transport in the BBB.
[0056] It will be appreciated that any of the aspects mentioned above can be applied individually or in combination with the aforementioned achievement.
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30/171 [0057] A method, for increasing CNS retention of a compound administered to a subject, in which the compound is coupled to an antibody that binds with low affinity to a BBB-R, so that retention in the CNS of the compound is increased, and in which the reduction of red blood cell levels in the subject upon administration of the antibody coupled to the compound to the subject is decreased or eliminated. In one aspect, BBB-R is selected from the group consisting of transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF receptor), protein 8 related to the low lipoprotein receptor density (LRP8), protein 1 related to the low density lipoprotein receptor (LRP1), glucose transporter 1 (Glutl) and growth factor similar to heparin-binding epidermal growth factor (HB-EGF). In another aspect, BBB-R is a human BBB-R. In one aspect, BBB-R is TfR. In another aspect, BBB-R is TfR, and the antibody does not inhibit TfR activity. In another aspect, BBB-R is TfR, and the antibody does not inhibit TfR activity to transferrin.
[0058] In another aspect, red blood cells are immature red blood cells. In another aspect, immature red blood cells are reticulocytes. In another aspect, the reduction in reticulocyte levels is accompanied by acute clinical symptoms. In another aspect, the method also comprises the stage of monitoring the subject by red blood cell depletion.
[0059] In another aspect, one or more properties of the antibody have been modified to reduce the impact of the antibody on reticulocyte levels and / or to reduce the severity or presence of acute clinical symptoms in the subject. In one aspect, the antibody's affinity for BBB-R is modified, that is, decreased. In another aspect, the effector function of the antibody's Fc region is modified. In one aspect, the effector function was reduced or
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31/171 eliminated in relation to the effector function of a wild type antibody of the same isotype. In another aspect, the effector function is reduced or eliminated by reducing glycosylation of the antibody. In another aspect, the glycosylation of the antibody is reduced by production of the antibody in an environment that does not allow for wild-type glycosylation. In one aspect, the antibody is produced in a non-mammalian cell production system. In another aspect, the antibody is produced synthetically. In another aspect, the glycosylation of the antibody is reduced by removing carbohydrate groups already present in the antibody. In another aspect, glycosylation of the antibody is reduced by modifying the antibody so that wild-type glycosylation does not occur. In another aspect, the antibody Fc region comprises a mutation at position 297, so that the wild-type asparagine residue at that position is replaced by another amino acid that interferes with glycosylation at that position. In another aspect, the effector function is reduced or eliminated by modifying the antibody isotype to an isotype that naturally has reduced or eliminated effector function.
[0060] In another aspect, the Fc region is modified to reduce or eliminate the effector function. In another aspect, the effector function is reduced or eliminated by at least one modification of the Fc region. In one aspect, the modification is a point mutation in the Fc region to impair binding to one or more Fc receptors selected from the following positions: 238, 239, 248, 249, 252, 254, 265, 268, 269, 270 , 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301,303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414 , 416, 419, 434, 435, 437, 438 and 439. In another aspect, the modification is elimination of some or all of the Fc regions. In another aspect, the effector function is reduced or eliminated by deletion of all or a portion of the Fc region, or by genetic elaboration of the antibody, so that it does not include a competent Fc region for effector function. On a
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32/171 aspect, the antibody is selected from a Fab or a single chain antibody.
[0061] In one aspect, the antibody's Fc and / or non-Fc region is modified to reduce or eliminate activation of the complement pathway by the antibody. In one aspect, the modification is a point mutation in the Fc region to impair binding to C1q, selected from the following positions: 270, 322, 329 and 321. In another aspect, the modification is elimination of some or all of the Fc regions. In another aspect, the complement activation function is reduced or eliminated by deletion of all or a portion of the Fc region, or by genetic elaboration of the antibody, so that it does not include an Fc region that engages in the complement pathway. In one aspect, the antibody is selected from a Fab or a single chain antibody. In another aspect, the non-Fc region of the antibody is modified to reduce or eliminate activation of the complement pathway by the antibody. In one aspect, the modification is a point mutation of the CH1 region to impair binding to C3. In one aspect, the point mutation is at position 132 (see, for example, Vidarte et a., (2001) J. BioL Chem. 276 (41): 3821738223).
[0062] In another aspect, the amount of dose and / or frequency of administration of the antibody is modulated to reduce the concentration of the antibody to which the red blood cells are exposed. In another aspect, the antibody is modified to comprise pH-sensitive BBB-R binding.
[0063] In another aspect, an additional compound is administered in addition to the antibody and the coupled compound. In one aspect, the additional compound is responsible for or contributes to the failure to reduce reticulocyte levels. In another aspect, the additional compound inhibits or prevents activation or activity of the complement pathway (see, for example, Mollnes and Kirschfink (2006) Molec. Immunol. 43: 107-121). In another aspect, the compound
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33/171 still protects reticulocytes from antibody-related depletion. In another aspect, the additional compound supports the growth, development or restoration of reticulocytes. In another aspect, the additional compound is selected from erythropoietin (EPO), an iron supplement, vitamin C, folic acid and vitamin B12. In another aspect, the additional compound is red blood cells or reticulocytes from the same subject. In another aspect, the additional compound is another subject's red blood cells or reticulocytes.
[0064] In another aspect, the compound is a neurological dysfunction drug. In another aspect, the compound is an imaging agent. In another aspect, the compound is labeled. In another aspect, the antibody is labeled. In another aspect, the antibody does not impair the binding of BBB-R to one or more of its native ligands. In another aspect, the antibody binds specifically to TfR, so that it does not inhibit the binding of TfR to transferrin. In another aspect, the compound is administered to a mammal. In another aspect, the mammal is a human. In another aspect, the mammal has neurological dysfunction. In another aspect, neurological dysfunction 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, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer and traumatic brain injury.
[0065] In another aspect, the increase in the CNS retention of the compound is measured in relation to the CNS retention of a compound coupled to a typical antibody that has no decreased affinity for BBB-R. In another aspect, the increase in CNS retention of the compound is measured as a ratio of the amount of the compound found in the CNS to the amount found in the serum at one or more points in time after administration. In another aspect, the increase in CNS retention results in
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34/171 a rate greater than 0.1% at one or more points in time after administration. In another aspect, the increase in CNS retention to the compound is measured in relation to the CNS exposure of a compound in the absence of a coupled antibody. In another aspect, the increase in CNS retention of the compound is measured by imaging. In another aspect, the increase in CNS retention of the compound is measured by an indirect reading, such as a modification of one or more physiological symptoms.
[0066] In another aspect, the antibody has an IC50 for BBB-R of about 1 nM to about 100 μΜ. In another aspect, the IC50 is about 5 nM to about 100 μΜ. In another aspect, the IC50 is about 50 nM to about 100 μΜ. In another aspect, the IC50 is about 100 nM to about 100 μΜ. In another aspect, the antibody has an BBB-R affinity of about 5 nM to about 50 μΜ. In another aspect, the antibody has an affinity for BBB-R from about 30 nM to about 30 μΜ. In another aspect, the antibody, when coupled to a compound, has an affinity for BBB-R from about 30 nM to about 1 μΜ. In another aspect, the antibody, when coupled to a compound, has an affinity for BBB-R from about 50 nM to about 1 μΜ. In another aspect, the antibody coupled to the complex binds specifically to TfR and has an affinity for TfR between the observed affinities for the anti-TíR ^A / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In another aspect, the antibody coupled to the complex binds specifically to TfR and has an affinity for TfR between the observed affinities for the anti-TfR ^D / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In another aspect, the antibody coupled to the complex specifically binds to TfR and has an IC50 for TfR among the IC50s observed for the anti-TfR ^A / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In another aspect, the antibody coupled to the complex binds specifically to TfR and has an IC50 for TfR among the IC50s observed for the anti-TfR ^D / BACE1 antibody and the anti
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TíR ^and / BACE1. In one aspect, the affinity of the anti-BBB-R or anti-BBBR / compound for the BBB-R is measured using Scatchard analysis. In another aspect, the affinity of anti-BBB-R or anti-BBB-R / compound for BBBR is measured using BIACORE analysis. In another aspect, the affinity of the anti-BBB-R or anti-BBB-R / compound for the BBB-R is measured with the use of a competition ELISA.
[0067] In another aspect, the dissociation half-life of the BBB-R antibody to which it specifically binds is about 30 seconds to about 30 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 20 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 10 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 5 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 3 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 2 minutes. In another aspect, the dissociation half-life is about 2 minutes. In another aspect, the dissociation half-life is 1 minute or less. In another aspect, the antibody coupled to the compound specifically binds to TfR and has a dissociation half-life for TfR between the dissociation half-lives observed for the anti-TfR ^A 7BACE1 antibody and the anti-TfR ^E / BACE1 antibody of their respective connections to TfR. In another aspect, the antibody coupled to the compound specifically binds to TfR and has a dissociation half-life for TfR between the dissociation half-lives observed for the anti-TfR ^D / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. their respective connections to TfR. In another aspect, the dissociation half-life of the anti-BBB-R or anti-BBB-R / compound for the BBB-R is measured using BIACORE analysis. In another aspect, the dissociation half-life of the anti-BBB-R or anti-BBB-R / compound for the BBBR is measured using a competition binding assay, such as a
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Competition ELISA.
[0068] In another aspect, the antibody coupled to the compound is administered in a therapeutic dose. In one aspect, the therapeutic dose is a dose that saturates the BBB-R to which the antibody specifically binds. In another aspect, the antibody coupled to the compound is administered at a dose and dose frequency that minimizes the interaction of red blood cells with the antibody coupled to the compound although it still facilitates the distribution of the compound through the BBB in the CNS at therapeutic levels.
[0069] In another aspect, the compound is covalently coupled to the antibody. In one aspect, the compound is linked to the antibody by a linker. In one aspect, the binder is cleavable. In another aspect, the binder is not cleavable. In another aspect, the compound is bound directly to the antibody. In one aspect, the antibody is a multispecific antibody and the compound optionally forms a portion of the multispecific antibody. In another aspect, the multispecific antibody comprises a first antigen binding site that binds to BBB-R and a second antigen binding site that binds to a brain antigen, beta-secretase 1 (BACE1), Abeta, receptor for epidermal growth factor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine repeat rich kinase 2 (LRRK2 ), parquin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), neurotrophin receptor p75 (p75NTR) caspase 6. In another aspect, the multispecific antibody binds both to TfR like BACE1. In another aspect, the multispecific antibody binds to both TfR and Abeta. In another aspect, the multispecific antibody is labeled. In another aspect, the compound is reversibly coupled to the antibody, so that the compound is released from the antibody with or after transport in the BBB.
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37/171 [0070] It will be appreciated that any of the aspects mentioned above can be applied individually or in combination with the aforementioned achievement.
[0071] In another embodiment, the invention provides a method for optimizing the pharmacokinetics and / or pharmacodynamics of a compound to be effective on a subject's CNS, characterized in that the compound is coupled to an antibody that binds with low affinity to a BBB-R, and the antibody is selected so that its affinity for BBB-R after coupling to the compound results in an amount of transport of the antibody conjugated to the compound through the BBB that optimizes the pharmacokinetics and / or pharmacodynamics of the compound in the CNS , wherein the reduction of red blood cell levels in the subject by administering the antibody coupled to the compound to the subject is decreased or eliminated. In one aspect, BBB-R is selected from the group consisting of transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF receptor), protein 8 related to the low lipoprotein receptor density (LRP8), protein 1 related to the low density lipoprotein receptor (LRP1), glucose transporter 1 (Glutl) and growth factor similar to heparin-binding epidermal growth factor (HB-EGF). In another aspect, BBB-R is a human BBB-R. In one aspect, BBB-R is TfR. In another aspect, BBB-R is TfR, and the antibody does not inhibit TfR activity. In another aspect, BBB-R is TfR, and the antibody does not inhibit TfR activity to transferrin.
[0072] In another aspect, red blood cells are immature red blood cells. In another aspect, immature red blood cells are reticulocytes. In another aspect, the reduction in reticulocyte levels is accompanied by acute clinical symptoms. In another aspect, the method also comprises the stage of monitoring the subject by depletion of blood cells
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38/171 red.
[0073] In another aspect, one or more properties of the antibody have been modified to reduce the impact of the antibody on reticulocyte levels and / or to reduce the severity or presence of acute clinical symptoms in the subject. In one aspect, the antibody's affinity for BBB-R is modified, that is, decreased. In another aspect, the effector function of the antibody's Fc region is modified. In one aspect, the effector function has been reduced or eliminated in relation to the effector function of a wild type antibody of the same isotype. In another aspect, the effector function is reduced or eliminated by reducing glycosylation of the antibody. In another aspect, the glycosylation of the antibody is reduced by production of the antibody in an environment that does not allow for wild-type glycosylation. In one aspect, the antibody is produced in a non-mammalian cell production system. In another aspect, the antibody is produced synthetically. In another aspect, the glycosylation of the antibody is reduced by removing carbohydrate groups already present in the antibody. In another aspect, glycosylation of the antibody is reduced by modifying the antibody so that wild-type glycosylation does not occur. In another aspect, the Fc region of the antibody comprises a mutation at position 297, so that the wild-type asparagine residue at that position is replaced by another amino acid that interferes with glycosylation at that position. In another aspect, the effector function is reduced or eliminated by modifying the antibody isotype to an isotype that naturally has reduced or eliminated effector function.
[0074] In another aspect, the Fc region is modified to reduce or eliminate the effector function. In another aspect, the effector function is reduced or eliminated by at least one modification of the Fc region. In one aspect, the modification is a point mutation in the Fc region to impair binding to one or more Fc receptors selected from the following positions: 238,
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39/171
239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301,303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438 and 439. In another aspect, the modification is elimination of some or all of the Fc regions. In another aspect, the effector function is reduced or eliminated by deletion of all or a portion of the Fc region, or by genetic elaboration of the antibody, so that it does not include a competent Fc region for effector function. In one aspect, the antibody is selected from a Fab or a single chain antibody.
[0075] In one aspect, the antibody's Fc and / or non-Fc region is modified to reduce or eliminate activation of the complement pathway by the antibody. In one aspect, the modification is a point mutation in the Fc region to impair binding to C1q, selected from the following positions: 270, 322, 329 and 321. In another aspect, the modification is elimination of some or all of the Fc regions. In another aspect, the complement activation function is reduced or eliminated by deletion of all or a portion of the Fc region, or by genetic elaboration of the antibody, so that it does not include an Fc region that engages in the complement pathway. In one aspect, the antibody is selected from a Fab or a single chain antibody. In another aspect, the non-Fc region of the antibody is modified to reduce or eliminate activation of the complement pathway by the antibody. In one aspect, the modification is a point mutation of the CH1 region to impair binding to C3. In one aspect, the point mutation is at position 132 (see, for example, Vidarte et a., (2001) J. Biol. Chem. 276 (41): 3821738223).
[0076] In another aspect, the amount of dose and / or frequency of administration of the antibody is modulated to reduce the concentration of the antibody to which the red blood cells are exposed. In another aspect, the
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40/171 antibody is modified to comprise pH sensitive BBB-R binding.
[0077] In another aspect, an additional compound is administered in addition to the antibody and the coupled compound. In one aspect, the additional compound is responsible for or contributes to the failure to reduce reticulocyte levels. In another aspect, the additional compound inhibits or prevents activation or activity of the complement pathway (see, for example, Mollnes and Kirschfink (2006) Molec. Immunol. 43: 107-121). In another aspect, the compound also protects reticulocytes from antibody-related depletion. In another aspect, the additional compound supports the growth, development or restoration of reticulocytes. In another aspect, the additional compound is selected from erythropoietin (EPO), an iron supplement, vitamin C, folic acid and vitamin B12. In another aspect, the additional compound is red blood cells or reticulocytes from the same subject. In another aspect, the additional compound is another subject's red blood cells or reticulocytes.
[0078] In another aspect, the compound is a neurological dysfunction drug. In another aspect, the compound is an imaging agent. In another aspect, the compound is labeled. In another aspect, the antibody is labeled. In another aspect, the antibody does not impair the binding of BBB-R to one or more of its native ligands. In another aspect, the antibody binds specifically to TfR, so that it does not inhibit the binding of TfR to transferrin. In another aspect, the BBB is in a mammal. In another aspect, the mammal is a human. In another aspect, the mammal has neurological dysfunction. In another aspect, neurological dysfunction 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, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer and traumatic brain injury. In another aspect, BBB is
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41/171 in a human.
[0079] In one aspect, optimization may include the generation of a series of antibody-compound complexes in which each antibody has a different affinity for BBB-R, and which assesses the pharmacokinetics and / or pharmacodynamics of each in the CNS. In another aspect, the optimization may be relative to a known pattern, such as, but not limited to, the pharmacokinetics and / or pharmacodynamics of the compound when introduced directly into the CNS or when introduced to the subject in the absence of a coupled anti-BBB-R antibody.
[0080] In another aspect, the antibody has an IC50 for BBB-R of about 1 nM to about 100 μΜ. In another aspect, the IC50 is about 5 nM to about 100 μΜ. In another aspect, the IC50 is about 50 nM to about 100 μΜ. In another aspect, the IC50 is about 100 nM to about 100 μΜ. In another aspect, the antibody has an BBB-R affinity of about 5 nM to about 50 μΜ. In another aspect, the antibody has an affinity for BBB-R from about 30 nM to about 30 μΜ. In another aspect, the antibody, when coupled to a compound, has an affinity for BBB-R from about 30 nM to about 1 μΜ. In another aspect, the antibody, when coupled to a compound, has an affinity for the BBB-R from about 50 nM to about 1 μΜ. In another aspect, the antibody coupled to the complex specifically binds TfR and has an affinity for TfR between the observed affinities for the anti-TfR ^A / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In another aspect, the antibody coupled to the complex binds specifically to TfR and has an affinity for TfR between the observed affinities for the anti-TfR ^D / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In another aspect, the antibody coupled to the complex specifically binds to TfR and has an IC50 for TfR among the IC50s observed for the anti-TfR ^A / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In another aspect, the antibody coupled to the
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42/171 complex binds specifically to TfR and has an IC50 for TfR among the IC50s observed for the anti-TfR antibody ^D / BACE1 and the antiTir ^e / BACE1 antibody. In one aspect, the affinity of the anti-BBB-R or anti-BBBR / compound for the BBB-R is measured using Scatchard analysis. In another aspect, the affinity of anti-BBB-R or anti-BBB-R / compound for BBBR is measured using BIACORE analysis. In another aspect, the affinity of the anti-BBB-R or anti-BBB-R / compound for the BBB-R is measured with the use of a competition ELISA.
[0081] In another aspect, the dissociation half-life of the BBB-R antibody to which it specifically binds is about 30 seconds to about 30 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 20 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 10 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 5 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 3 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 2 minutes. In another aspect, the dissociation half-life is about 2 minutes. In another aspect, the dissociation half-life is 1 minute or less. In another aspect, the antibody coupled to the compound specifically binds to TfR and has a dissociation half-life for TfR between the dissociation half-lives observed for the anti-TfR ^A 7BACE1 antibody and the anti-TfR ^E / BACE1 antibody of their respective connections to TfR. In another aspect, the antibody coupled to the compound specifically binds to TfR and has a dissociation half-life for TfR between the dissociation half-lives observed for the anti-TfR ^D / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. their respective connections to TfR. In another aspect, the dissociation half-life of the anti-BBB-R or anti-BBB-R / compound for the BBB-R is measured using BIACORE analysis. In another aspect, the
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43/171 Dissociation half-life of the anti-BBB-R or anti-BBB-R / compound for the BBBR is measured using a competition binding assay, such as a competition ELISA.
[0082] In another aspect, the antibody coupled to the compound is administered in a therapeutic dose. In one aspect, the therapeutic dose is a dose that saturates the BBB-R to which the antibody specifically binds. In another aspect, the antibody coupled to the compound is administered at a dose and dose frequency that minimizes the interaction of red blood cells with the antibody coupled to the compound although it still facilitates the distribution of the compound through the BBB in the CNS at therapeutic levels.
[0083] In another aspect, the compound is covalently coupled to the antibody. In one aspect, the compound is linked to the antibody by a linker. In one aspect, the binder is cleavable. In another aspect, the binder is not cleavable. In another aspect, the compound is bound directly to the antibody. In one aspect, the antibody is a multispecific antibody and the compound optionally forms a portion of the multispecific antibody. In another aspect, the multispecific antibody comprises a first antigen binding site that binds to BBB-R and a second antigen binding site that binds to a brain antigen, beta-secretase 1 (BACE1), Abeta, receptor for epidermal growth factor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine repeat rich kinase 2 (LRRK2 ), parquin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), neurotrophin receptor p75 (p75NTR) caspase 6. In another aspect, the multispecific antibody binds both to TfR like BACE1. In another aspect, the multispecific antibody binds to both TfR and Abeta. In another aspect, the multispecific antibody is labeled. In another aspect, the compound is reversibly coupled to the
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44/171 antibody, so that the compound is released from the antibody with or after transport in the BBB.
[0084] It will be appreciated that any of the aspects mentioned above can be applied individually or in combination with the aforementioned achievement.
[0085] In another embodiment, the invention provides a method for treating neurological dysfunction in a mammal, which comprises treating the mammal with an antibody that binds to a BBB-R and is coupled to a compound, in which the antibody has been selected to have low affinity for BBBR and thereby improve the absorption in the CNS of the antibody and the coupled compound, and in which the reduction of red blood cell levels in the subject by administering the antibody coupled to the compound to the subject is decreased or eliminated. In one aspect, BBB-R is selected from the group consisting of transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF receptor), protein 8 related to the low lipoprotein receptor density (LRP8), protein 1 related to the low density lipoprotein receptor (LRP1), glucose transporter 1 (Glutl) and growth factor similar to heparin-binding epidermal growth factor (HB-EGF). In another aspect, BBB-R is a human BBB-R. In one aspect, BBB-R is TfR. In another aspect, BBB-R is TfR, and the antibody does not inhibit TfR activity. In another aspect, BBB-R is TfR, and the antibody does not inhibit TfR activity to transferrin.
[0086] In another aspect, red blood cells are immature red blood cells. In another aspect, immature red blood cells are reticulocytes. In another aspect, the reduction in reticulocyte levels is accompanied by acute clinical symptoms. In another aspect, the method also comprises the stage of monitoring the subject by depletion of blood cells
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45/171 red.
[0087] In another aspect, one or more properties of the antibody have been modified to reduce the impact of the antibody on reticulocyte levels and / or to reduce the severity or presence of acute clinical symptoms in the subject. In one aspect, the antibody's affinity for BBB-R is modified, that is, decreased. In another aspect, the effector function of the antibody's Fc region is modified. In one aspect, the effector function has been reduced or eliminated in relation to the effector function of a wild type antibody of the same isotype. In another aspect, the effector function is reduced or eliminated by reducing glycosylation of the antibody. In another aspect, the glycosylation of the antibody is reduced by production of the antibody in an environment that does not allow for wild-type glycosylation. In one aspect, the antibody is produced in a non-mammalian cell production system. In another aspect, the antibody is produced synthetically. In another aspect, the glycosylation of the antibody is reduced by removing carbohydrate groups already present in the antibody. In another aspect, glycosylation of the antibody is reduced by modifying the antibody so that wild-type glycosylation does not occur. In another aspect, the Fc region of the antibody comprises a mutation at position 297, so that the wild-type asparagine residue at that position is replaced by another amino acid that interferes with glycosylation at that position. In another aspect, the effector function is reduced or eliminated by modifying the antibody isotype to an isotype that naturally has reduced or eliminated effector function.
[0088] In another aspect, the Fc region is modified to reduce or eliminate the effector function. In another aspect, the effector function is reduced or eliminated by at least one modification of the Fc region. In one aspect, the modification is a point mutation in the Fc region to impair binding to one or more Fc receptors selected from the following positions: 238,
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46/171
239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301,303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438 and 439. In another aspect, the modification is elimination of some or all of the Fc regions. In another aspect, the effector function is reduced or eliminated by deletion of all or a portion of the Fc region, or by genetic elaboration of the antibody, so that it does not include a competent Fc region for effector function. In one aspect, the antibody is selected from a Fab or a single chain antibody.
[0089] In another aspect, the amount of dose and / or frequency of administration of the antibody is modulated to reduce the concentration of the antibody to which the red blood cells are exposed. In another aspect, the antibody is modified to comprise pH-sensitive BBB-R binding.
[0090] In another aspect, an additional compound is administered in addition to the antibody and the coupled compound. In one aspect, the additional compound is responsible for or contributes to the failure to reduce reticulocyte levels. In another aspect, the additional compound inhibits or prevents activation or activity of the complement pathway (see, for example, Mollnes and Kirschfink (2006) Molec. Immunol. 43: 107-121). In another aspect, the compound also protects reticulocytes from antibody-related depletion. In another aspect, the additional compound supports the growth, development or restoration of reticulocytes. In another aspect, the additional compound is selected from erythropoietin (EPO), an iron supplement, vitamin C, folic acid and vitamin B12. In another aspect, the additional compound is red blood cells or reticulocytes from the same subject. In another aspect, the additional compound is another subject's red blood cells or reticulocytes.
[0091] In another aspect, the compound is a neurological dysfunction drug. In another aspect, the compound is a
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47/171 images. In another aspect, the compound is labeled. In another aspect, the antibody is labeled. In another aspect, the antibody does not impair the binding of BBB-R to one or more of its native ligands. In another aspect, the antibody binds specifically to TfR, so that it does not inhibit the binding of TfR to transferrin. In one aspect, the mammal is a human. In another aspect, the mammal has neurological dysfunction. In another aspect, neurological dysfunction 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, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer and traumatic brain injury.
[0092] In one aspect, treatment results in a reduction or elimination of dysfunction symptoms. In another aspect, treatment results in improvement of neurological dysfunction.
[0093] In another aspect, the antibody has an IC50 for BBB-R of about 1 nM to about 100 μΜ. In another aspect, the IC50 is about 5 nM to about 100 μΜ. In another aspect, the IC50 is about 50 nM to about 100 μΜ. In another aspect, the IC50 is about 100 nM to about 100 μΜ. In another aspect, the antibody has an BBB-R affinity of about 5 nM to about 50 μΜ. In another aspect, the antibody has an affinity for BBB-R from about 30 nM to about 30 μΜ. In another aspect, the antibody, when coupled to a compound, has an affinity for BBB-R from about 30 nM to about 1 μΜ. In another aspect, the antibody, when coupled to a compound, has an affinity for the BBB-R from about 50 nM to about 1 μΜ. In another aspect, the antibody coupled to the complex binds specifically to TfR and has an affinity for TfR between the observed affinities for the anti-TfR ^A 7BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In another aspect, the antibody coupled to the complex binds specifically
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48/171 to TfR and has an affinity for TfR between the observed affinities for the anti-TfR ^D / BACE1 antibody and the anti-TfR ^ BACEI antibody. In another aspect, the antibody coupled to the complex specifically binds to TfR and has an IC50 for TfR among the IC50s observed for the anti-TfR ^A / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In another aspect, the antibody bound to the complex specifically binds the TfR and has an IC50 for TfR observed between the IC50 for anti-TfR antibody ^D / BACE1 and antiTíR antibody ^and / BACE1. In one aspect, the affinity of the anti-BBB-R or anti-BBBR / compound for the BBB-R is measured using Scatchard analysis. In another aspect, the affinity of anti-BBB-R or anti-BBB-R / compound for BBBR is measured using BIACORE analysis. In another aspect, the affinity of the anti-BBB-R or anti-BBB-R / compound for the BBB-R is measured with the use of a competition ELISA.
[0094] In another aspect, the dissociation half-life of the BBB-R antibody to which it specifically binds is about 30 seconds to about 30 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 20 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 10 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 5 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 3 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 2 minutes. In another aspect, the dissociation half-life is about 2 minutes. In another aspect, the dissociation half-life is 1 minute or less. In another aspect, the antibody coupled to the compound specifically binds to TfR and has a dissociation half-life for TfR between the dissociation half-lives observed for the anti-TfR ^A / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. their respective connections to TfR. In another aspect, the antibody coupled to the compound binds
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49/171 specifically to TfR and has a dissociation half-life for TfR between the dissociation half-lives observed for the anti-TfR antibody ^D / BACE1 and the anti-TfR antibody ^E / BACE1 from their respective TfR bonds. In another aspect, the dissociation half-life of the anti-BBB-R or anti-BBB-R / compound for the BBB-R is measured using BIACORE analysis. In another aspect, the dissociation half-life of the anti-BBB-R or anti-BBB-R / compound for the BBBR is measured using a competition binding assay, such as a competition ELISA.
[0095] In another aspect, the antibody coupled to the compound is administered in a therapeutic dose. In one aspect, the therapeutic dose is a dose that saturates the BBB-R to which the antibody specifically binds. In another aspect, the antibody coupled to the compound is administered at a dose and dose frequency that minimizes the interaction of red blood cells with the antibody coupled to the compound although it still facilitates the distribution of the compound through the BBB in the CNS at therapeutic levels.
[0096] In another aspect, the compound is covalently coupled to the antibody. In one aspect, the compound is linked to the antibody by a linker. In one aspect, the binder is cleavable. In another aspect, the binder is not cleavable. In another aspect, the compound is bound directly to the antibody. In one aspect, the antibody is a multispecific antibody and the compound optionally forms a portion of the multispecific antibody. In another aspect, the multispecific antibody comprises a first antigen binding site that binds to BBB-R and a second antigen binding site that binds to a brain antigen, beta-secretase 1 (BACE1), Abeta, receptor for epidermal growth factor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine repeat rich kinase 2 (LRRK2 ), parquina, presenilin 1, presenilin 2, gamma secretase, receptor 6 of
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50/171 death (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR) caspase 6. In another aspect, the multispecific antibody binds to both TfR and BACE1. In another aspect, the multispecific antibody binds to both TfR and Abeta. In another aspect, the multispecific antibody is labeled. In another aspect, the compound is reversibly coupled to the antibody, so that the compound is released from the antibody with or after transport in the BBB.
[0097] It will be appreciated that any of the aspects mentioned above can be applied individually or in combination with the aforementioned achievement.
[0098] In another embodiment, the invention provides a method for enhancing the safety in a subject of an antibody that binds to a BBB-R, which comprises modifying one or more properties of the antibody, so that administration of the antibody decreases or eliminate the reduction in red blood cell levels in the subject observed after administration of the unmodified antibody. In one aspect, BBB-R is selected from the group consisting of transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF receptor), protein 8 related to the low lipoprotein receptor density (LRP8), protein 1 related to the low density lipoprotein receptor (LRP1), glucose transporter 1 (Glutl) and growth factor similar to heparin-binding epidermal growth factor (HB-EGF). In another aspect, BBB-R is a human BBB-R. In one aspect, BBB-R is TfR. In another aspect, BBB-R is TfR, and the antibody does not inhibit TfR activity. In another aspect, BBB-R is TfR, and the antibody does not inhibit TfR activity to transferrin.
[0099] In another aspect, red blood cells are immature red blood cells. In another aspect, immature red blood cells are reticulocytes. In another aspect, the reduction in reticulocyte levels is
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51/171 accompanied by acute clinical symptoms.
[00100] In another aspect, one or more properties of the antibody have been modified to reduce the impact of the antibody on reticulocyte levels and / or to reduce the severity or presence of acute clinical symptoms in the subject. In one aspect, the antibody's affinity for BBB-R is modified, that is, decreased. In another aspect, the modification of antibody affinity is measured against a wild type antibody of the same isotype having no modified (i.e., decreased) affinity for BBB-R. In another aspect, the effector function of the antibody's Fc region is modified. In one aspect, the effector function has been reduced or eliminated in relation to the effector function of a wild type antibody of the same isotype. In another aspect, the effector function is reduced or eliminated by reducing glycosylation of the antibody. In another aspect, the glycosylation of the antibody is reduced by production of the antibody in an environment that does not allow for wild-type glycosylation. In one aspect, the antibody is produced in a non-mammalian cell production system. In another aspect, the antibody is produced synthetically. In another aspect, the glycosylation of the antibody is reduced by removing carbohydrate groups already present in the antibody. In another aspect, glycosylation of the antibody is reduced by modifying the antibody so that wild-type glycosylation does not occur. In another aspect, the antibody Fc region comprises a mutation at position 297, so that the wild-type asparagine residue at that position is replaced by another amino acid that interferes with glycosylation at that position. In another aspect, the effector function is reduced or eliminated by modifying the antibody isotype to an isotype that naturally has reduced or eliminated effector function.
[00101] In another aspect, the Fc region is modified to reduce or eliminate the effector function. In another aspect, the effector function is reduced or eliminated by at least one modification of the Fc region. On a
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52/171 aspect, the modification is a point mutation in the Fc region to impair binding to one or more Fc receptors selected from the following positions: 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301,303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438 and 439. In another aspect, the modification is elimination of some or all of the Fc regions. In another aspect, the effector function is reduced or eliminated by deletion of all or a portion of the Fc region, or by genetic elaboration of the antibody, so that it does not include a competent Fc region for effector function. In one aspect, the antibody is selected from a Fab or a single chain antibody.
[00102] In one aspect, the antibody's Fc and / or non-Fc region is modified to reduce or eliminate activation of the complement pathway by the antibody. In one aspect, the modification is a point mutation in the Fc region to impair binding to C1q, selected from the following positions: 270, 322, 329 and 321. In another aspect, the modification is elimination of some or all of the Fc regions. In another aspect, the complement activation function is reduced or eliminated by deletion of all or a portion of the Fc region, or by genetic elaboration of the antibody, so that it does not include an Fc region that engages in the complement pathway. In one aspect, the antibody is selected from a Fab or a single chain antibody. In another aspect, the non-Fc region of the antibody is modified to reduce or eliminate activation of the complement pathway by the antibody. In one aspect, the modification is a point mutation of the CH1 region to impair binding to C3. In one aspect, the point mutation is at position 132 (see, for example, Vidarte et al., (2001) J. Biol. Chem. 276 (41): 3821738223).
[00103] In another aspect, the dose quantity and / or
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The frequency of administration of the antibody is modulated to reduce the concentration of the antibody to which the red blood cells are exposed. In another aspect, the antibody is modified to comprise pH-sensitive BBB-R binding.
[00104] In another aspect, the antibody is coupled to a therapeutic compound. In another aspect, the compound is a neurological dysfunction drug. In another aspect, the compound is an imaging agent. In another aspect, the compound is labeled. In another aspect, the antibody is labeled. In another aspect, the antibody does not impair the binding of BBB-R to one or more of its native ligands. In another aspect, the antibody binds specifically to TfR, so that it does not inhibit the binding of TfR to transferrin. In another aspect, the BBB is in a mammal. In another aspect, the mammal is a human. In another aspect, the mammal has neurological dysfunction. In another aspect, neurological dysfunction 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's syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer and traumatic brain injury. In another aspect, the BBB is in a human.
[00105] In another aspect, the antibody has an IC50 for the BBB-R of about 1 nMa about 100 μΜ. In another aspect, the IC50 is from about 5 nM to about 100 μΜ. In another aspect, the IC50 is about 50 nM to about 100 μΜ. In another aspect, the IC50 is about 100 nM to about 100 μΜ. In another aspect, the antibody has an BBB-R affinity of about 5 nM to about 50 μΜ. In another aspect, the antibody has an affinity for BBB-R from about 30 nM to about 30 μΜ. In another aspect, the antibody, when coupled to a compound, has an affinity for
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54/171 the BBB-R of about 30 nM to about 1 μΜ. In another aspect, the antibody, when coupled to a compound, has an affinity for the BBB-R from about 50 nM to about 1 μΜ. In another aspect, the antibody coupled to the complex binds specifically to TfR and has an affinity for TfR between the observed affinities for the anti-TfR ^A / BACE1 antibody and the antiTir ^e / BACE1 antibody. In another aspect, the antibody coupled to the complex specifically binds to TfR and has an affinity for TfR between the observed affinities for the anti-TfR antibody ^c 7BACE1 and the anti-TfR ^E / BACE1 antibody. In another aspect, the antibody coupled to the complex specifically binds to TfR and has an IC50 for TfR among the IC50s observed for the anti-TfR ^A / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In another aspect, the antibody coupled to the complex specifically binds to TfR and has an IC50 for TfR among the IC50s observed for the anti-TfR ^D / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In one aspect, the affinity of the anti-BBB-R or anti-BBBR / compound for the BBB-R is measured using Scatchard analysis. In another aspect, the affinity of anti-BBB-R or anti-BBB-R / compound for BBBR is measured using BIACORE analysis. In another aspect, the affinity of the anti-BBB-R or anti-BBB-R / compound for the BBB-R is measured with the use of a competition ELISA.
[00106] In another aspect, the dissociation half-life of the BBB-R antibody to which it specifically binds is about 30 seconds to about 30 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 20 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 10 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 5 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 3 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 2 minutes. In another aspect, the sock
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55/171 dissociation life is about 2 minutes. In another aspect, the dissociation half-life is 1 minute or less. In another aspect, the antibody coupled to the compound specifically binds to TfR and has a dissociation half-life for TfR between the dissociation half-lives observed for the anti-TfR ^A ./BACE1 antibody and the anti-TfR ^E / antibody. BACE1 of their respective TfR links. In another aspect, the antibody coupled to the compound specifically binds to TfR and has a dissociation half-life for TfR between the dissociation half-lives observed for the anti-TfR ^D / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. their respective connections to TfR. In another aspect, the dissociation half-life of the anti-BBB-R or anti-BBB-R / compound for the BBB-R is measured using BIACORE analysis. In another aspect, the dissociation half-life of the anti-BBB-R or anti-BBB-R / compound for the BBBR is measured using a competition binding assay, such as a competition ELISA.
[00107] In another aspect, the antibody is selected from an antibody panel based on the affinity of the selected antibody. In another aspect, the antibody is genetically engineered to have the desired affinity. In one aspect, the antibody is generated using any protein genetic engineering methodology known in the art including, but not limited to, phage display, yeast display, random mutagenesis and site-directed mutagenesis.
[00108] In another aspect, the antibody coupled to the compound is administered in a therapeutic dose. In one aspect, the therapeutic dose is a dose that saturates the BBB-R to which the antibody specifically binds. In another aspect, the antibody coupled to the compound is administered at a dose and a dose frequency that minimizes the interaction of red blood cells with the antibody coupled to the compound although it still facilitates the distribution of the compound through the BBB in the CNS at levels
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56/171 therapeutic.
[00109] In another aspect, the compound is covalently coupled to the antibody. In one aspect, the compound is linked to the antibody by a linker. In one aspect, the binder is cleavable. In another aspect, the binder is not cleavable. In another aspect, the compound is bound directly to the antibody. In one aspect, the antibody is a multispecific antibody and the compound optionally forms a portion of the multispecific antibody. In another aspect, the multispecific antibody comprises a first antigen binding site that binds to BBB-R and a second antigen binding site that binds to a brain antigen. In another aspect, the cerebral antigen is selected from the group consisting of: beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER2), Tau , apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine repeat rich kinase 2 (LRRK2), parchin, presenilin 1, presenilin 2, gamma secretase, receptor 6 of death (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), interleukin receptor 6 (IL6R), TNF receptor 1 (TNFR1), beta 1 interleukin and caspase 6. In another aspect, the multispecific antibody binds both to TfR like BACE1. In another aspect, the multispecific antibody binds to both TfR and Abeta. In another aspect, the multispecific antibody is labeled. In another aspect, the compound is reversibly coupled to the antibody, so that the compound is released from the antibody with or after transport in the BBB.
[00110] It will be appreciated that any of the aspects mentioned above can be applied individually or in combination with the aforementioned achievement.
[00111] In another embodiment, the invention provides a method for producing an antibody useful for transporting a compound via the BBB
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57/171 with enhanced safety, characterized by the fact that it comprises selecting a specific antibody for a blood brain barrier receptor (BBBR) that has a low desirable affinity for BBB-R, and modifying one or more properties of the antibody, so that administration of the antibody decreases or eliminates the reduction in the levels of red blood cells in the subject, seen by administration of an unmodified antibody. In one aspect, BBB-R is selected from the group consisting of transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF receptor), protein 8 related to the low lipoprotein receptor density (LRP8), protein 1 related to the low density lipoprotein receptor (LRP1), glucose transporter 1 (Glutl) and growth factor similar to heparin-binding epidermal growth factor (HB-EGF). In another aspect, BBB-R is a human BBB-R. In one aspect, BBB-R is TfR. In another aspect, BBB-R is TfR, and the antibody does not inhibit TfR activity. In another aspect, BBB-R is TfR, and the antibody does not inhibit TfR activity to transferrin.
[00112] In another aspect, red blood cells are immature red blood cells. In another aspect, immature red blood cells are reticulocytes. In another aspect, the reduction in reticulocyte levels is accompanied by acute clinical symptoms.
[00113] In another aspect, one or more properties of the antibody have been modified to reduce the impact of the antibody on reticulocyte levels and / or to reduce the severity or presence of acute clinical symptoms in the subject. In one aspect, the antibody's affinity for BBB-R is modified, that is, decreased. In another aspect, the modification of antibody affinity is measured against a wild type antibody of the same isotype having no modified (i.e., decreased) affinity for BBB-R. In another aspect, the effector function of the antibody's Fc region is modified. On a
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58/171 aspect, the effector function was reduced or eliminated in relation to the effector function of a wild type antibody of the same isotype. In another aspect, the effector function is reduced or eliminated by reducing glycosylation of the antibody. In another aspect, the glycosylation of the antibody is reduced by production of the antibody in an environment that does not allow for wild-type glycosylation. In one aspect, the antibody is produced in a non-mammalian cell production system. In another aspect, the antibody is produced synthetically. In another aspect, the glycosylation of the antibody is reduced by removing carbohydrate groups already present in the antibody. In another aspect, glycosylation of the antibody is reduced by modifying the antibody so that wild-type glycosylation does not occur. In another aspect, the antibody Fc region comprises a mutation at position 297, so that the wild-type asparagine residue at that position is replaced by another amino acid that interferes with glycosylation at that position. In another aspect, the effector function is reduced or eliminated by modifying the antibody isotype to an isotype that naturally has reduced or eliminated effector function.
[00114] In another aspect, the Fc region is modified to reduce or eliminate the effector function. In another aspect, the effector function is reduced or eliminated by at least one modification of the Fc region. In one aspect, the modification is a point mutation in the Fc region to impair binding to one or more Fc receptors selected from the following positions: 238, 239, 248, 249, 252, 254, 265, 268, 269, 270 , 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301,303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414 , 416, 419, 434, 435, 437, 438 and 439. In another aspect, the modification is elimination of some or all of the Fc regions. In another aspect, the effector function is reduced or eliminated by deletion of all or a portion of the Fc region, or by genetic elaboration of the antibody, so that it does not include a competent Fc region for effector function. On a
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59/171 aspect, the antibody is selected from a Fab or a single chain antibody.
[00115] In one aspect, the antibody's Fc and / or non-Fc region is modified to reduce or eliminate activation of the complement pathway by the antibody. In one aspect, the modification is a point mutation in the Fc region to impair binding to C1q, selected from the following positions: 270, 322, 329 and 321. In another aspect, the modification is elimination of some or all of the Fc regions. In another aspect, the complement activation function is reduced or eliminated by deletion of all or a portion of the Fc region, or by genetic elaboration of the antibody, so that it does not include an Fc region that engages in the complement pathway. In one aspect, the antibody is selected from a Fab or a single chain antibody. In another aspect, the non-Fc region of the antibody is modified to reduce or eliminate activation of the complement pathway by the antibody. In one aspect, the modification is a point mutation of the CH1 region to impair binding to C3. In one aspect, the point mutation is at position 132 (see, for example, Vidarte et al, (2001) J. BioL Chem. 276 (41): 3821738223).
[00116] In another aspect, the amount of dose and / or frequency of administration of the antibody is modulated to reduce the concentration of the antibody to which the red blood cells are exposed. In another aspect, the antibody is modified to comprise pH-sensitive BBB-R binding.
[00117] In another aspect, the antibody is coupled to a therapeutic compound. In another aspect, the compound is a neurological dysfunction drug. In another aspect, the compound is an imaging agent. In another aspect, the compound is labeled. In another aspect, the antibody is labeled. In another aspect, the antibody does not harm the
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60/171 binding of BBB-R to one or more of its native ligands. In another aspect, the antibody binds specifically to TfR, so that it does not inhibit the binding of TfR to transferrin. In another aspect, the BBB is in a mammal. In another aspect, the mammal is a human. In another aspect, the mammal has neurological dysfunction. In another aspect, neurological dysfunction 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, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer and traumatic brain injury. In another aspect, the BBB is in a human.
[00118] In another aspect, the antibody has an IC50 for BBB-R of about 1 nM to about 100 μΜ. In another aspect, the IC50 is about 5 nM to about 100 μΜ. In another aspect, the IC50 is about 50 nM to about 100 μΜ. In another aspect, the IC50 is about 100 nM to about 100 μΜ. In another aspect, the antibody has an BBB-R affinity of about 5 nM to about 50 μΜ. In another aspect, the antibody has an affinity for BBB-R from about 30 nM to about 30 μΜ. In another aspect, the antibody, when coupled to a compound, has an affinity for BBB-R from about 30 nM to about 1 μΜ. In another aspect, the antibody, when coupled to a compound, has an affinity for the BBB-R from about 50 nM to about 1 μΜ. In another aspect, the antibody coupled to the complex binds specifically to TfR and has an affinity for TfR between the observed affinities for the anti-TfR ^A / BACE1 antibody and the antiTir ^e / BACE1 antibody. In another aspect, the antibody coupled to the complex binds specifically to TfR and has an affinity for TfR between the observed affinities for the anti-TfR ^D / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In another aspect, the antibody coupled to the complex binds specifically
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61/171 to TfR and has an IC50 for TfR among the IC50s observed for the anti-TfR ^A / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In another aspect, the antibody coupled to the complex specifically binds to TfR and has an IC50 for TfR among the IC50s observed for the anti-TfR ^D / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In one aspect, the affinity of the anti-BBB-R or anti-BBBR / compound for the BBB-R is measured using Scatchard analysis. In another aspect, the affinity of anti-BBB-R or anti-BBB-R / compound for BBBR is measured using BIACORE analysis. In another aspect, the affinity of the anti-BBB-R or anti-BBB-R / compound for the BBB-R is measured with the use of a competition ELISA.
[00119] In another aspect, the dissociation half-life of the BBB-R antibody to which it specifically binds is about 30 seconds to about 30 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 20 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 10 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 5 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 3 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 2 minutes. In another aspect, the dissociation half-life is about 2 minutes. In another aspect, the dissociation half-life is 1 minute or less. In another aspect, the antibody coupled to the compound specifically binds to TfR and has a dissociation half-life for TfR between the dissociation half-lives observed for the anti-TfR ^A 7BACE1 antibody and the anti-TfR ^E / BACE1 antibody of their respective connections to TfR. In another aspect, the antibody coupled to the compound specifically binds to TfR and has a dissociation half-life for TfR between the dissociation half-lives observed for the anti-TfR ^D / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. their respective connections to TfR. In another
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62/171 aspect, the dissociation half-life of the anti-BBB-R or anti-BBB-R / compound for the BBB-R is measured using BIACORE analysis. In another aspect, the dissociation half-life of the anti-BBB-R or anti-BBB-R / compound for the BBBR is measured using a competition binding assay, such as a competition ELISA.
[00120] In another aspect, the antibody is selected from an antibody panel based on the affinity of the selected antibody. In another aspect, the antibody is genetically engineered to have the desired affinity. In one aspect, the antibody is generated using any protein genetic engineering methodology known in the art including, but not limited to, phage display, yeast display, random mutagenesis and site-directed mutagenesis.
[00121] In another aspect, the antibody coupled to the compound is administered in a therapeutic dose. In one aspect, the therapeutic dose is a dose that saturates the BBB-R to which the antibody specifically binds. In another aspect, the antibody coupled to the compound is administered at a dose and dose frequency that minimizes the interaction of red blood cells with the antibody coupled to the compound although it still facilitates the distribution of the compound through the BBB in the CNS at therapeutic levels.
[00122] In another aspect, the compound is covalently coupled to the antibody. In one aspect, the compound is linked to the antibody by a linker. In one aspect, the binder is cleavable. In another aspect, the binder is not cleavable. In another aspect, the compound is bound directly to the antibody. In one aspect, the antibody is a multispecific antibody and the compound optionally forms a portion of the multispecific antibody. In another aspect, the multispecific antibody comprises a first antigen binding site that binds to BBB-R and a second antigen binding site
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63/171 antigen that binds to a brain antigen, beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER2), tau, apolipoprotein E4 (ApoE4 ), alphasinuclein, CD20, huntingtin, prion protein (PrP), leucine 2 repeat rich kinase (LRRK2), parquine, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), neurotrophin receptor p75 (p75NTR) caspase 6. In another aspect, the multispecific antibody binds to both TfR and BACE1. In another aspect, the multispecific antibody binds to both TfR and Abeta. In another aspect, the multispecific antibody is labeled. In another aspect, the compound is reversibly coupled to the antibody, so that the compound is released from the antibody with or after transport in the BBB.
[00123] It will be appreciated that any of the aspects mentioned above can be applied individually or in combination with the aforementioned achievement.
[00124] In another embodiment, the invention provides an antibody that binds to a blood-brain barrier receptor (BBB-R), in which the antibody's affinity to BBB-R is about 5 nM to about 50 μΜ, and wherein one or more properties of the antibody have been modified to reduce at least one unwanted side effect on red blood cells. In one aspect, BBB-R is selected from the group consisting of transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF receptor), protein 8 related to the low lipoprotein receptor density (LRP8), protein 1 related to the low density lipoprotein receptor (LRP1), glucose transporter 1 (Glutl) and growth factor similar to heparin-binding epidermal growth factor (HB-EGF). In another aspect, BBB-R is a human BBB-R. In one aspect, BBB-R is TfR. In another aspect, BBB-R is TfR, and the antibody does not inhibit
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64/171 TfR activity. In another aspect, BBB-R is TfR and the antibody does not inhibit TfR binding to transferrin.
[00125] In another aspect, red blood cells are immature red blood cells. In another aspect, immature red blood cells are reticulocytes. In another aspect, the reduction in reticulocyte levels is accompanied by acute clinical symptoms.
[00126] In another aspect, one or more properties of the antibody have been modified to reduce the impact of the antibody on reticulocyte levels and / or to reduce the severity or presence of acute clinical symptoms in the subject. In one aspect, the antibody's affinity for BBB-R is modified, that is, decreased. In another aspect, the modification of antibody affinity is measured against a wild type antibody of the same isotype having no modified (i.e., decreased) affinity for BBB-R. In another aspect, the effector function of the antibody's Fc region is modified. In one aspect, the effector function has been reduced or eliminated in relation to the effector function of a wild type antibody of the same isotype. In another aspect, the effector function is reduced or eliminated by reducing glycosylation of the antibody. In another aspect, the glycosylation of the antibody is reduced by production of the antibody in an environment that does not allow for wild-type glycosylation. In one aspect, the antibody is produced in a non-mammalian cell production system. In another aspect, the antibody is produced synthetically. In another aspect, the glycosylation of the antibody is reduced by removing carbohydrate groups already present in the antibody. In another aspect, glycosylation of the antibody is reduced by modifying the antibody so that wild-type glycosylation does not occur. In another aspect, the antibody Fc region comprises a mutation at position 297, so that the wild-type asparagine residue at that position is replaced by another amino acid that interferes with glycosylation at that position. In another aspect, the effector function
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65/171 is reduced or eliminated by modifying the antibody's isotype to an isotype that naturally has reduced or eliminated effector function.
[00127] In another aspect, the Fc region is modified to reduce or eliminate the effector function. In another aspect, the effector function is reduced or eliminated by at least one modification of the Fc region. In one aspect, the modification is a point mutation in the Fc region to impair binding to one or more Fc receptors selected from the following positions: 238, 239, 248, 249, 252, 254, 265, 268, 269, 270 , 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301,303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414 , 416, 419, 434, 435, 437, 438 and 439. In another aspect, the modification is elimination of some or all of the Fc regions. In another aspect, the effector function is reduced or eliminated by deletion of all or a portion of the Fc region, or by genetic elaboration of the antibody, so that it does not include a competent Fc region for effector function. In one aspect, the antibody is selected from a Fab or a single chain antibody.
[00128] In one aspect, the antibody's Fc and / or non-Fc region is modified to reduce or eliminate activation of the complement pathway by the antibody. In one aspect, the modification is a point mutation in the Fc region to impair binding to C1q, selected from the following positions: 270, 322, 329 and 321. In another aspect, the modification is elimination of some or all of the Fc regions. In another aspect, the complement activation function is reduced or eliminated by deletion of all or a portion of the Fc region, or by genetic elaboration of the antibody, so that it does not include an Fc region that engages in the complement pathway. In one aspect, the antibody is selected from a Fab or a single chain antibody. In another aspect, the non-Fc region of the antibody is modified to reduce or eliminate activation of the complement pathway by the antibody. On a
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66/171 aspect, the modification is a point mutation of the CH1 region to impair binding to C3. In one aspect, the point mutation is at position 132 (see, for example, Vidarte et al., (2001) J. Biol. Chem. 276 (41): 3821738223).
[00129] In another aspect, the antibody is coupled to a therapeutic compound. In another aspect, the compound is a neurological dysfunction drug. In another aspect, the compound is an imaging agent. In another aspect, the compound is labeled. In another aspect, the antibody is labeled. In another aspect, the antibody does not impair the binding of BBB-R to one or more of its native ligands. In another aspect, the antibody binds specifically to TfR, so that it does not inhibit the binding of TfR to transferrin. In another aspect, the BBB is in a mammal. In another aspect, the mammal is a human. In another aspect, the mammal has neurological dysfunction. In another aspect, neurological dysfunction 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, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer and traumatic brain injury. In another aspect, the BBB is in a human.
[00130] In another aspect, the antibody has an IC50 for BBB-R of about 30 nM to about 30 μΜ. In another aspect, the antibody, when coupled to a compound, has an affinity for BBB-R from about 30 nM to about 1 μΜ. In another aspect, the antibody coupled to the complex binds specifically to TfR and has an affinity for TfR between the observed affinities for the anti-TfR ^A / BACE1 antibody and the antiTfR ^E / BACE1 antibody. In another aspect, the antibody coupled to the complex binds specifically to TfR and has an affinity for TfR between the affinities
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67/171 observed for anti-TfR ^D / BACE1 antibody and anti-TfR ^E / BACE1 antibody. In another aspect, the antibody coupled to the complex specifically binds to TfR and has an IC50 for TfR among the IC50s observed for the anti-TfR ^A / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In another aspect, the antibody coupled to the complex specifically binds to TfR and has an IC50 for TfR among the IC50s observed for the anti-TfR ^D / BACE1 antibody and the anti-TfR ^E / BACE1 antibody. In one aspect, the affinity of the anti-BBB-R or anti-BBBR / compound for the BBB-R is measured using Scatchard analysis. In another aspect, the affinity of anti-BBB-R or anti-BBB-R / compound for BBBR is measured using BIACORE analysis. In another aspect, the affinity of the anti-BBB-R or anti-BBB-R / compound for the BBB-R is measured with the use of a competition ELISA.
[00131] In another aspect, the dissociation half-life of the BBB-R antibody to which it specifically binds is about 30 seconds to about 30 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 20 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 10 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 5 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 3 minutes. In another aspect, the dissociation half-life is about 30 seconds to about 2 minutes. In another aspect, the dissociation half-life is about 2 minutes. In another aspect, the dissociation half-life is 1 minute or less. In another aspect, the antibody coupled to the compound specifically binds to TfR and has a dissociation half-life for TfR between the dissociation half-lives observed for the anti-TfR ^A 7BACE1 antibody and the anti-TfR ^E / BACE1 antibody of their respective connections to TfR. In another aspect, the antibody coupled to the compound binds specifically to TfR and has a dissociation half-life for TfR between the
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68/171 dissociation half-lives observed for the anti-TfR ^D / BACE1 antibody and the anti-TfR ^E / BACE1 antibody from their respective TfR binding. In another aspect, the dissociation half-life of the anti-BBB-R or anti-BBB-R / compound for the BBB-R is measured using BIACORE analysis. In another aspect, the dissociation half-life of the anti-BBB-R or anti-BBB-R / compound for the BBBR is measured using a competition binding assay, such as a competition ELISA.
[00132] In another aspect, the antibody is selected from an antibody panel based on the affinity of the selected antibody. In another aspect, the antibody is genetically engineered to have the desired affinity. In one aspect, the antibody is generated using any protein genetic engineering methodology known in the art including, but not limited to, phage display, yeast display, random mutagenesis and site-directed mutagenesis.
[00133] In another aspect, a compound is covalently coupled to the antibody. In one aspect, the compound is linked to the antibody by a linker. In one aspect, the binder is cleavable. In another aspect, the binder is not cleavable. In another aspect, the compound is bound directly to the antibody. In one aspect, the antibody is a multispecific antibody and the compound optionally forms a portion of the multispecific antibody. In another aspect, the multispecific antibody comprises a first antigen binding site that binds to BBB-R and a second antigen binding site that binds to a brain antigen. In another aspect, the cerebral antigen is selected from the group consisting of: beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER2), Tau , apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), kinase rich in leucine 2 repeat (LRRK2), parchin, presenilin 1, presenilin 2, gamma
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69/171 secretase, death 6 receptor (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), interleukin receptor 6 (IL6R), TNF receptor 1 (TNFR1), interleukin beta 1 and caspase 6 In another aspect, the multispecific antibody binds both TfR and BACE1. In another aspect, the multispecific antibody binds to both TfR and Abeta. In another aspect, the multispecific antibody is labeled. In another aspect, the compound is reversibly coupled to the antibody, so that the compound is released from the antibody with or after transport in the BBB.
[00134] It will be appreciated that any of the aspects mentioned above can be applied individually or in combination with the aforementioned achievement.
[00135] In another embodiment, the invention provides the use of an antibody that binds with low affinity to a BBB-R and that does not reduce the levels of red blood cells, for the manufacture of a drug for the treatment of a neurological dysfunction. Any of the low affinity anti-BBB-R antibodies described previously or any of the low affinity anti-BBB-R antibodies described elsewhere can be used in the method.
[00136] In another embodiment, the invention provides an antibody that binds with low affinity to a BBB-R and that does not reduce the levels of red blood cells, for use in treating a neurological dysfunction. Any of the low affinity anti-BBB-R antibodies described previously or any of the low affinity anti-BBB-R antibodies described elsewhere can be used in the method.
[00137] In another embodiment, the invention provides a method for transporting a therapeutic compound, such as a neurological dysfunction drug, across the blood-brain barrier which comprises exposing the anti-BBB-R antibody coupled to a neurological dysfunction drug in a manner
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70/171 that the antibody carries the neurological dysfunction drug coupled to it through the blood-brain barrier, and the antibody does not reduce red blood cell levels.
[00138] The invention additionally provides a method for treating a neurological dysfunction in a mammal which comprises treating the mammal with a multispecific antibody that binds to both a blood brain barrier (BBB-R) receptor and a brain antigen, wherein the anti-BBB-R antibody has been selected to have a low affinity for BBB-R and thereby improves brain anti-brain antigen absorption in the brain, and the administration of the antibody does not decrease red blood cell levels.
[00139] The invention additionally provides a method for treating a disease or dysfunction associated with or caused by high levels of red blood cells in a subject, characterized in that it comprises administering an anti-TfR antibody that comprises at least partial effector function for the subject. In one aspect, the administration step is at a dose and / or frequency of the calibrated dose to minimize acute clinical symptoms of antibody administration.
[00140] It is understood that any of the previously mentioned methods and compositions of the invention can be combined with each other and / or with the additional aspects of the invention described in the specification in the present application.
Brief Description of the Figures [00141] Figures 1A to 1E represent the results of experiments evaluating the affinities of anti-transferrin receptor (TfR ”) and anti-TfR / beta-secretase (“ BACE1 ”) variants for TfR, as well as concentrations of the antibody and Αβ1-40 after administration to mice, as described in Example 1. The results of the competitive ELISA assay
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71/171 in Figure 1A shows that the anti-DTR / BACE1 and anti-TfR variants have different affinities for DTR. Figures 1B and 1D show, respectively, the mean serum and brain antibody concentrations in wild-type mice after a single intravenous injection of 50 mg / kg of control IgG, anti-BACE1, or a variant of anti-TfR / BACE1 (n = 6 per group). Figures 10 and 1E show the concentrations in plasma and brain and Αβι-40, respectively, in these same treated mice, as a marker of the activity of the injected antibody.
[00142] Figure 2A is a schematic representation of the maturation of red blood cells (RBC) in the bone marrow, showing progression from pro-erythroblast (Pro-EB), to basophilic erythroblast (Baso-EB), polychromatic erythroblast (Poly- EB), orthochromatic erythroblast (OrtoEB) and finally for reticulocytes. Reticulocytes are released from the bone marrow into the circulation where they mature for RBCs. During the maturation stages in the bone marrow, erythroid precursors synthesize the hemoglobin protein containing iron, which requires a concomitant increase in TfR expression. Transferrin receptors are dispersed with the cessation of hemoglobin synthesis and cell proliferation as cells mature in the reticulocyte stage, so that mature RBCs do not express TfR. The relative number of TfR present in each RBC cell maturation stage is shown in the graph at the top of the figure, based on data from lacpetta et at, Biochim. Blophys. Acta 687: 204-210 (1982). Figures 2B and 2C represent the results of experiments evaluating the impact of administration of anti-TfR and anti-TfR / BACE1 on the reticulocytes in mice, as described in Example 2A. Figure 2B represents the results of experiments that test the impact of anti-TfR ^D , antiTfR ^D / BACE1 or control IgG, administered intravenously on the percentage of immature reticulocytes in the whole blood fraction of
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72/171 wild-type mice at 1 hour post-dose (n = 6 per group). Figure 2C represents the results of experiments that test the impact of antiTíR ^a / BACE1, antiTfR ^D / BACE1 or control IgG, administered intravenously to count the total reticulocytes in the whole blood of wild type mice in 24 hours or 7 days post -dose (n = 6 per group). All data are shown as mean ± SEM. Figures 2D and 2E represent concentrations of Abeta-Mo in wild type mice after a single intravenous injection of 50 mg / kg of control IgG or 5 mg / kg, 25 mg / kg or 50 mg / kg of anti-TfR injections. ^D / BACE1 (Figure 2D) or antiTíR ^a / BACE1 (Figure 2E) (n = 6 per group). Figures 2F to 2H represent the results of experiments evaluating the pharmacokinetics of anti-TfR ^A / BACE1 and anti-TfR ^D / BACE1 compared to the control at dose levels of 5 mg / kg, 25 mg / kg or 50 mg / kg. Figure 2F provides measurements of the concentration of antibodies in the brain at the indicated time points. Figure 2G provides measurements of the plasma antibody concentration at the indicated time points. Figure 2H provides measurements of Abeta plasma levels at the indicated time points.
[00143] Figures 3A to 3E represent the results of experiments evaluating the impact of eliminating the effector function (Figures 3A to 3C) or the elimination of the complement function (Figures 3D and 3E) in the depletion of reticulocytes by various anti-TfR antibodies , as described in Example 2B. Total reticulocyte counts in whole blood are shown from wild mice (Figures 3A and 3C), Εογ " ^Λ mice (B6.129P2-Fcer1gtm1 Rav N12) (Figure 3B), or C3" mice (Figure 3D), 24 hours after intravenous injection of antibody at the indicated dose, compared to the IgG control group (n = 6 per group). Figure 3E represents the results of experiments evaluating the effect of the complement system deficiency on the observed depletion
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73/171 previously of reticulocytes by anti-TfR. Wild or C3 knockout mice were administered intravenously with 50 mg / kg of a control IgG or anti-TfR ^D / control IgG mixture (n = 6 per group).
[00144] Figures 4A and 4B represent the results of in vitro experiments evaluating the induction of antibody-dependent cell-mediated cytotoxicity (ADCC) (Figure 4A) or complement-dependent cytotoxicity (CDC) (Figure 4B) by anti-TfR ^A , anti-TfR ^A / BACE1 or control IgG in mouse erythroleukemic blasts at an antibody concentration range, as described in Example 2B.
[00145] Figures 5A to 5C show the results of experiments evaluating whether the elimination of Fc binding or BACE1 binding impacts reticulocyte depletion by monospecific or bispecific anti-TfR antibodies, as described in Example 2C. Total reticulocyte counts are shown for wild type mice (n = 6 per group) 24 hours after intravenous injection of the indicated F (ab ') ₂ or control IgG (Figures 5A and 5B) or bispecific antibody (Figure 5C).
[00146] Figures 6A to 6C represent the results of experiments evaluating the impact of reduced affinity for TfR on reticulocyte depletion and TfR expression in the brain, as described in Example 3. Figures 6A and 6B represent the total reticulocyte counts in wild-type mice 24 hours after intravenous injection of the indicated anti-TfR / BACE1 antibody, compared to control IgG. Figure 6C shows the quantification of the level of TfR expression in the brain by Western blot of total mouse brain lysates, 4 days after an intravenous injection of control IgG, anti-TfR ^Ã / BACE1 or anti “TfR ^D / BACE1 in indicated dose (n = 3 per group). Quantification of TfR expression has been normalized for actin and data are shown as mean ± SEM.
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74/171 [00147] Figures 7A to 7C represent the results of experiments evaluating whether treatment with TfR antibody affected the blood-brain barrier permeability, as described in Example 4. Wild-type mice were administered intravenously at 50 mg / kg of Control IgG or 25 mg / kg of each of the co-injected antibody combinations. The mean antibody absorption in the brain 24 hours after intravenous injection was measured using a generic human Fc ELISA (Figure 7-A) or a BACE1 ectodomain ELISA (Figure 7B). Figure 7C shows a quantification of Αβι.40 concentrations in the mouse brain after intravenous injection of control IgG or co-injection of antibodies (n = 6 per group).
[00148] Figures 8A to 8F show the results of experiments evaluating the impact of multiple doses of anti-TfR ^D / BACE1 on reticulocyte levels in treated mice, as described in Example 5. Wild type mice were dosed intravenously once per week with 25 mg / kg of control IgG or anti-TfR ^D / BACE1. Figures 8A and 8B, respectively, represent antibody concentrations observed in the plasma and brain at 24 hours, 4 days and 7 days after two or four doses of antibody. It should be noted that the scale of the Y axis in Figure 8A is in μΜ, while the scale of the Y axis in Figure 8B is in nM. The corresponding mean concentrations of Αβ-ι. ^ In the plasma (Figure 8C) and in the brain (Figure 8D) were also measured. Figure 8E shows the total reticulocyte count in mice 24 hours after the second and fourth doses, and 7 days after the fourth dose of control IgG or anti-TfR ^D / BACE1. Figure 8F shows a graph depicting the results of a quantification of the level of TfR expression in the brain by Western blot of total mouse brain lysates after 4 weekly doses of control IgG or antiTfR ^D / BACE1. Quantification of TfR expression was normalized for actin
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75/171 and data are shown as mean ± SEM.
[00149] Figures 9A to 9B and 10A to 10D represent the results of experiments evaluating the impact of a less effector antiTIR / BACE1 antibody on the erythrocyte subpopulations in blood and bone marrow in mice. Different populations of Ter119 positive erythrocyte strains, both in blood (Figure 9A) and in bones (Figure 9B), are differentiated by their TfR expression and cell size (as determined by frontal dispersion profile) using cytometry of flow (Paniga et a /., PLoS One 6, 9 (2011)). Subsets of Ter119 positive cells in the bone marrow were defined as large EryA ^, initial basophilic erythroblasts positive for TfR, small EryB ^, polychromatic erythroblasts positive for TfR, mature EryC- TfR negative erythrocytes. Figures 9C and 9D show a temporal evolution of the Ter119 positive erythrocyte population (reticulocytes and red blood cells; 9C) and TfR (9D) positive reticulocytes in the blood after dosing with antiTfR ^D / BACE1, compared to the control IgG (n = 6 / group). Figures 10A to 10D provide graphs of the quantification of different subpopulations of erythrocytes (EryA, EryB, EryC) in the bone marrow after anti-TfR ^D / BACE1 or control IgG measurement (n = 6 / group).
[00150] Figures 11A to 11B and 12A to 12D represent the results of experiments analyzing the impact of an effector function of an anti-TfR / BACE1 antibody on the erythrocyte populations in the blood and bone marrow in mice. Figures 11A to 11B show the quantification of Ter119 positive erythrocyte populations (Figure 11 A) and TfR positive reticulocyte populations (Figure 11B) in the blood after antiTfR ^A / BACE1 (Fc-) and anti-TfR ^D / BACE1 ( Fc-) less effector, and anti-TfR ^D / BACE1 (Fc +) with complete effector function, or control IgG measurement (n = 6 / group). Figures 12A to 12D provide the quantification of erythrocyte subpopulations
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76/171 (Ter119 positive erythrocyte lineage in Figure 12A; EryA in Figure 12B; EryB in Figure 12C; and EryC in Figure 12D) in the bone marrow after dosage of less effector and anti-TfR ^A / BACE1 (Fc-) anti-TfR ^D ./BACE1 (Fc-), antiTfR ^D / BACE1 (Fc +) with complete effector function, or control IgG measurement (n = 6 / group).
[00151] Figures 13A to B and Figures 14A to B represent the results of experiments evaluating the impact of the state of effector function on ADCC activity of anti-human TfR (anti-hTFR ”) antibodies in a human erythroblast cell line or primary bone marrow mononuclear cells, as described in Example 7.
[00152] Figures 15A-B represent the amino acid sequences of the light and heavy chain of the anti-BACE1 clone YW412.8, obtained from a virgin species from the natural diversity phage display library and mature affinity forms of YW412 .8. Figure 15A represents the sequence alignments of the light variable (VL) (SEQ ID NOs. 1-6). Figure 13B represents the sequence alignments of the heavy variable (VH) (SEQ ID NOs. 7-8). In both figures, the HVR sequences for each clone are indicated by the regions in boxes, with the first box indicating HVR-L1 (Figure 15A) or HVR-H1 (Figure 15B), the second box indicating HVR-L2 (Figure 15A ) or HVR-H2 (Figure 15B), and the third box indicating HVR-L3 (Figure 15A) or HVR-H3 (Figure 15B).
[00153] Figures 16A-B represent the amino acid sequences of the light and heavy chains of the Fab 12 clone of the antiBACE1 antibody, obtained from a virgin species of a phage display library of synthetic diversity and forms of mature Fab affinity 12. Figure 16A represents the light chain sequence alignments (SEQ ID NOs. 9-12). Figure 16B represents the heavy chain sequence alignments (SEQ ID NO. 13). In both figures, the HVR sequences for
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77/171 each clone is indicated by regions in boxes, with the first box indicating HVR-L1 (Figure 16A) or HVR-H1 (Figure 16B), the second box indicating HVR-L2 (Figure 16A) or HVR-H2 (Figure 16B), and the third box indicating HVR-L3 (Figure 16A) or HVR-H3 (Figure 16B).
[00142] Figures 17A-B represent the heavy chain (Figure 17A; SEQ ID NO. 14 and light chain (Figure 17B; SEQ ID NO. 15) of an exemplary anti-Abeta antibody.
Detailed Description of the Hnvention's Achievements
L Definitions [00143] The '' blood-brain barrier 'or' 'BBB' refers to the physiological barrier between the peripheral circulation and the brain and spinal cord (ie the CNS) which is formed by tight junctions within the endothelial plasma membranes capillaries of the brain, creating a strong barrier that restricts the transport of molecules in the brain, even very small molecules such as urea (60 Daltons). The blood-brain barrier within the brain, the blood-spinal cord barrier within the spinal cord and the blood-retinal barrier within the retina are contiguous capillary barriers within the CNS and in the present application are collectively referred to as a blood-brain barrier or BBB. BBB also encompasses the blood-brain barrier (choroid plexus) where the barrier is comprised of ependymal cells instead of capillary endothelial cells.
[00144] The "central nervous system" or "CNS" refers to the complex of nerve tissues that control bodily functions, and includes the brain and spinal cord.
[00145] A "blood-brain barrier receptor" (abbreviated as "BBB-R" in this application) is a transmembrane receptor protein expressed in brain endothelial cells that is capable of transporting molecules across the blood-brain barrier. BBB Examples
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R include, but are not limited to: transferrin receptor (TFR), insulin receptor, insulin-like growth factor receptor (IGF-R), low-density lipoprotein receptors including without limitation lipoprotein receptor-related protein 1 low density (LRP1) and protein 8 related to the low density lipoprotein receptor (LRP8), glucose transporter 1 (Glutl) and growth factor similar to heparin-binding epidermal growth factor (HB-EGF). An exemplary BBB-R in the present application is transferrin receptor (TfR).
[00146] The "transferrin receptor" is a transmembrane glycoprotein (with a molecular weight of approximately 180,000) composed of two disulfide-linked subunits (each with an apparent molecular weight of about 90,000) involved in the absorption of iron in vertebrates. In one embodiment, the TfR in the present application is human TfR which comprises the amino acid sequence as presented in Schneider et al. Nature 311: 675 - 678 (1984), for example.
[00147] A neurological dysfunction, "as used in this application", refers to a disease or disorder that affects the CNS and / or that has an etiology in the CNS. Exemplary CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, eye disease or dysfunction, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, seizure, behavioral disorders, lysosomal storage disease. For the purposes of the present application, the CNS will be understood to include the eye, which is normally separated from the rest of the body by the hemato-retinal barrier. Specific examples of neurological dysfunctions 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, tauopathies
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79/171 including, but not limited to, Alzheimer's disease and supranuclear palsy), prionic diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler- Scheinker, chronic weakness and fatal familial insomnia), bulbar paralysis, motor neuron disease and heterodegenerative nervous system disorders (including, but not limited to, Canavan's disease, Huntington's disease, neuronal lipofuscinosis, Alexander disease, Tourette's syndrome, Menkes' curly hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, Lafora's disease, Rett's syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome and Unverricht-Lundborg syndrome), dementia (including, but not limited to) if limited to, Pick's disease and spinocerebellar ataxia), cancer (for example, CNS, including brain metastases resulting from cancer somewhere in the body).
[00148] A "neurological dysfunction drug" is a drug or therapeutic agent that treats one or more neurological dysfunction (s). Neurological dysfunction drugs of the invention include, but are not limited to, antibodies, peptides, proteins, natural ligands of one or more target CNS (s), modified versions of natural ligands of one or more target CNS (s), aptamers, acids inhibitory nuclei (i.e., small inhibitory RNAs (siRNA) and short-clip RNAs (shRNA)), ribozymes, and small molecules or active fragments of any of the aforementioned. Exemplary neurological dysfunction drugs of the invention are described in the present application and include, but are not limited to: antibodies, aptamers, proteins, peptides, inhibitory nucleic acids and small molecules and active fragments of any of the aforementioned which are both themselves and specifically recognize and / or act under (ie, inhibit, activate or detect) a CNS antigen or target molecule like, but not
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80/171 limited to, amyloid precursor protein or portions thereof, amyloid beta, beta-secretase, gamma-secretase, tau, alpha-synuclein parquine, huntingtin, DR6, presenilin, ApoE, glioma or other cancer markers of the CNS and neurotrophins . Non-limiting examples of drugs for neurological disorders and the disorders they can be used to treat are given in Table 1 below:
Table
Non-Limiting Examples of Neurological Dysfunction Drugs and Their
Corresponding dysfunctions that can be used to treat
Damn it Neurological dysfunction Anti-BACE1 Antibody Acute and chronic Alzheimer's brain injury, stroke Anti-Abeta Antibody Alzheimer's disease Anti-Tau Antibody Alzheimer's disease, taupathies Neurotrophin Stroke, acute brain injury, spinal cord injury Brain-derived neurotrophic factor (BDNF), fibroblast growth factor 2 (FGF-2) Chronic brain injury (Neurogenesis) Antiepidermal growth factor (EGFR) receptor-antibody Brain cancer Neural factor derived from the glial cell line (GDNF) Parkinson's disease Brain-derived neurotrophic factor(BDNF) Amyotrophic lateral sclerosis,depression Lysosomal enzyme Lysosomal storage disorders of the brain Ciliary neurotrophic factor (CNTF) Amyotrophic lateral sclerosis Neuregulin-1 Schizophrenia Anti-HER2 antibody (for example, Brain metastasis from cancer
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Damn it Neurological dysfunction trastuzumab, pertuzumab, etc.) HER2-positive Anti-VEGF antibody (for example, bevacizumab) Newly diagnosed or recurrent glioblastoma, recurrent malignant glioma, brain metastasis
[00149] An "imaging agent" is a compound that has one or more properties that allow its presence and / or location to be detected directly or indirectly. Examples of such imaging agents include proteins and small molecule compounds that incorporate a labeled component that allows detection.
[00150] A "CNS antigen" or "brain antigen" is an antigen expressed on the CNS, including the brain, that can be targeted with an antibody or a small molecule. Examples of such antigens include, but are not limited to: beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER2), tau, apolipoprotein E4 (ApoE4 ), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine 2 repeat rich kinase (LRRK2), parquine, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP ), p75 neurotrophin receptor (p75NTR), interleukin receptor 6 (IL6R), TNF receptor 1 (TNFR1), beta 1 interleukin and caspase 6. In one embodiment, the antigen is BACE1.
[00151] The term "BACE1", as used in this application, refers to any native beta-secretase 1 (also called amyloid precursor protein dividing enzyme 1 of the β site, membrane-associated aspartic protease 2, memapsin 2, aspartyl protease 2 or Asp2) from any source of vertebrates, including mammals, such as primates (eg, humans) and rodents (eg, mice and rats), unless otherwise indicated. The term encompasses unprocessed entire BACE1,
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82/171 as well as any form of BACE1 that results from processing in the cell. The term also encompasses naturally occurring BACE1 variants, for example, splicing variants or allelic variants. The amino acid sequence of an exemplary BACE1 polypeptide is the sequence for human BACE1, isoform A as reported in Vassar et al., Science 286: 735-741 (1999), which is integrally incorporated into the present application as a reference. There are several other isoforms of human BACE1 including isoforms B, C and D. See UniProtKB / Swiss-Prot Entry P56817, which is integrally incorporated into the present application as a reference.
[00152] The terms '' anti-beta-secretase antibody ”, '' anti-BACE1 antibody”, 'an antibody that binds to beta-secretase' and 'an antibody that binds to BACE1' refer to an antibody which is able to bind to BACE1 with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting BACE1. In one embodiment, the degree of binding of an anti-BACE1 antibody to an unrelated non-BACE1 protein is less than about 10% of the antibody's binding to BACE1 as measured, for example, by a radioimmunoassay (RIA). In certain embodiments, an antibody that binds to BACE1 has a dissociation constant (Kd) <1μΜ, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM or <0.001 nM ( for example, 1O ^'8 M or less, e.g., 10' ³ M to 10 ^"13 M, e.g., 1O ^'& 1O M' M ^i3). In certain embodiments, an antiBACE1 antibody binds to an epitope of BACE1 that is conserved between BACE1 of different species and isoforms. In one embodiment, an antibody is provided that binds to the BACE1 epitope bound by the anti-BACE1 antibody YW412.8.31. In other embodiments, an antibody is provided that binds to an exosite within BACE1 located in the catalytic domain of BACE1. In one embodiment, an antibody is provided that competes with the peptides identified in Kornacker et al., Biochem. 44: 11567-11573 (2005), which is
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83/171 fully incorporated into the present application as a reference (i.e., peptides 1,2,3,1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 2 to 12, 3 to 12, 4 to 12, 5 to 12, 6 to 12, 7 to 12, 8 to 12, 9 to 12, 10 to 12, 4, 5, 6, 5 to 10, 5 to 9, mixed, Y5A, P6A , Y7A, F8A, I9A, P10A and L11 A) for connection to BACE1. Exemplary BACE1 antibody sequences are shown in Figure 15A-B and Figure 16A-B. An exemplary antibody in the present application comprises the variable domains of the YW412.8.31 antibody (for example, as in Figures 15A-B).
[00149] A "native sequence" protein in the present application refers to a protein that comprises the amino acid sequence of a protein found in nature, including naturally occurring variants of the protein. The term as used in the present application includes the protein as isolated from a natural source or as produced recombinantly.
[00150] The term "antibody" in this application is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies) formed by at least two intact antibodies and antibody fragments, as long as they exhibit the desired biological activity.
[00151] Antibody fragments ”in the present application comprise a portion of an intact antibody that maintains the ability to bind to the antigen. Examples of antibody fragments are well known in the art (see, for example, Nelson, MAbs (2010) 2 (1): 7783) and include, but are not limited to Fab, Fab ', F (ab') ₂ , and Fv fragments; diabodies; linear antibodies, single chain antibody molecules that include, but are not limited to, single chain variable fragments (scFv), light fusions and / or heavy chain antigen binding domains with or without a linker (and optionally in tandem ); and binding molecules of
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84/171 monospecific or multispecific antigens formed from antibody fragments (including, but not limited to multispecific antibodies constructed from multiple variable domains lacking in Fc regions).
[00152] The term '' monoclonal antibody 'as used in the present application refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants that may appear during the production of the monoclonal antibody, these variants are generally present in smaller quantities. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant in the antigen. In addition, for their specificities, monoclonal antibodies are advantageous, as they are not contaminated by other immunoglobulins. The "monoclonal" modifier indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be interpreted as a requirement for antibody production by any specific method. For example, monoclonal antibodies to be used according to the present invention can be produced by the hybridoma method, first described by Kohler et al., Nature, 256: 495 (1975), or they can be produced by recombinant DNA methods ( see, for example, US patent 4,816,567). "Monoclonal antibodies" can also be isolated from antibody phage libraries using the techniques described in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Blol. , 222: 581-597 (1991), for example. Specific examples of monoclonal antibodies in the present application include chimeric antibodies, antibodies
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85/171 humanized and human antibodies, including antigen binding fragments thereof.
[00153] The monoclonal antibodies in the present application specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequences in the antibodies derived from a specific species or belonging to a class or subclass specific antibody, while the rest of the chain (s) is (are) identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibody, as well as fragments of these antibodies, as long as they exhibit the desired biological activity (US patent 4,816,567; Morrison et at., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). Chimeric antibodies of interest in the present application include "primatized" antibodies that comprise variable domain antigen binding sequences derived from a non-human primate (for example, Old World Monkey, such as baboon, rhesus monkey or cynomolgus) and sequences of human constant region (US patent 5,693,780).
[00154] "Humanized" forms of non-human antibodies (eg, murine) are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Most humanized antibodies are human immunoglobulins (receptor antibody), in which residues from a hypervariable region of the receptor are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit or non-primate. that has the desired specificity, affinity and capacity. In some cases, residues from the human immunoglobulin framework (FR) region are replaced by corresponding non-human residues. In addition, humanized antibodies
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86/171 can comprise residues that are not found in the recipient antibody or donor antibody. These modifications are still made to refine the performance of the antibody. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, where all or substantially all hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all FRs are those of a sequence human immunoglobulin, except for RF replacement (s) as noted above. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin. For additional details, see Jones etal., Nature 321: 522-525 (1986); Riechmann et al, Nature 332: 323-329 (1988); and Presta, Cure Op. Struct. Biol. 2: 593-596 (1992).
[00155] A "human antibody", as described in the present application, is one that comprises an amino acid sequence structure that corresponds to an amino acid sequence structure of an antibody obtained from a human B cell, and includes fragments of antigen binding of human antibodies. These antibodies can be identified or manufactured by a variety of techniques, including, but not limited to: production by transgenic animals (for example, mice) that are capable, through immunization, of producing human antibodies in the absence of the production of endogenous immunoglobulin ( see, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and US patents 591,669, 5,589,369 and 5,545,807)); selection from phage libraries expressing human antibodies or human antibody fragments (see, for example, McCafferty et al, Nature 348: 552-553 (1990); Johnson et al, Current Opinion in Structural
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Biology 3: 564-571 (1993); Clackson et al., Nature, 352.624-628 (1991): Marks et al., J. Mol. Biol. 222: 581-597 (1991); Griffith et al., EMBO J. 12: 725-734 (1993); US patents. 5,565,332 and 5,573,905); generation through in vitro activated B cells (see US patents 5,567,610 and 5,229,275); and isolation from hybridomas that produce human antibody.
[00156] A ‘‘ multispecific antibody ”in the present application is an antibody that has binding specificities by at least two different epitopes. Exemplary multispecific antibodies can bind to both a BBB-R and a brain antigen. Multispecific antibodies can be prepared as whole antibodies or antibody fragments (for example, bispecific F (ab ') 2 antibodies). Antibodies genetically engineered with two, three or more (for example, four) functional antigen binding sites are also contemplated (see, for example, US patent application 2002/0004587 A1, Miller et al.). Multispecific antibodies can be prepared as whole antibodies or as fragments of antibodies.
[00157] The antibodies in the present application include "variant amino acid sequences" with antigen binding or altered biological activity. Examples of such amino acid changes include antibodies with increased affinity for antigen (e.g., 'mature affinity' antibodies), and antibodies with altered Fc region, if present, for example, with altered antibody-dependent cell cytotoxicity and / or dependent cytotoxicity complement (CDC) (see, for example, WO 00/42072. Presta, L. and WO 99/51642, Iduosogie et al.); and / or increased or decreased serum half-life (see, for example, WO 00/42072, Presta, L).
[00158] A ‘‘ modified affinity variant ’has one or more hypervariable regions replaced or structure residues of a
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88/171 parental antibody (for example, a chimeric, humanized or human parental antibody) that alters (increases or decreases) affinity. A convenient way to generate these substitutional variants uses phage display. Briefly, several hypervariable region sites (for example, sites 6 to 7) are mutated to generate all possible amino acid substitutions at each site. The variant antibodies thus generated are displayed in a monovalent manner from filamentous phage particles, as fusions for the gene III product of M13 packaged within each particle. The displayed phage variants are then selected for their biological activities (for example, binding affinity). In order to identify candidate hypervariable region sites for modifications, alanine scan mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify points of contact between the antibody and its target. These contact wastes and neighboring wastes are candidates for substitution according to the techniques developed in this application. Once these variants are generated, the panel of variants is subjected to selection and antibodies with altered affinity can be selected for further development.
[00159] A "pH sensitive variant antibody" is a variant antibody that has a different binding affinity for a target antigen at a first pH than it has for the target antigen at a different pH. As a non-limiting example, an anti-TfR antibody of the invention can be selected or genetically engineered to have pH sensitive binding to TfR, so that it desirably binds with low affinity (as described in the present application) to the TfR cell surface in plasma at pH 7.4, but by internalization in an endosomal compartment,
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89/171 quickly dissociates from TfR at a relatively lower pH (pH 5.5 to 6.0); this dissociation can protect the antibody from antigen-mediated clearance, and increase the amount of antibody that is either distributed to the CNS or recycled via the BBB - in both cases, the effective concentration of the antibody is increased over an anti- TfR that does not understand this pH sensitivity (see, for example, ChaparroRiggers et at. J. Biol. Chem. 287 (14): 11090-11097; Igawa et al., Nature Biotechnol. 28 (11): 1203-1208) . The desired combination of affinities at a serum pH and the pH of the endosomal compartment can be easily determined by a specific BBB-R and compound conjugated by a person skilled in the art.
[00160] The antibody in the present application can be conjugated to a "heterologous molecule", for example to increase half-life or stability, or otherwise, to improve the antibody. For example, the antibody can be attached to a variety of non-proteinaceous polymers, for example, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. Antibody fragments, such as Fab ', attached to one or more PEG molecules are an exemplary embodiment of the invention. In another example, the heterologous molecule is a therapeutic compound or a visualization agent (i.e., a detectable marker), and the antibody is being used to transport that heterologous molecule through the BBB. Examples of heterologous molecules include, but are not limited to, a chemical compound, a peptide, a polymer, a lipid, a nucleic acid and a protein.
[00161] The antibody in the present application can be a "glycosylation variant", so that any carbohydrate attached to the Fc region, if present, is altered, both modified in the presence / absence, and modified in type. For example, antibodies with a carbohydrate structure
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Mature 90/171 which are devoid of fucose bound to an Fc region of the antibody are described in US patent application 2003/0157108 (Presta, L). See also US patent 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with a divided N-acetylglucosamine (GlcNAc) in the carbohydrate bound to an Fc region of the antibody are referred to in WO 2003/011878, JeanMairet et al., And US patent 6,602,684, Umana et al. Antibodies with at least one galactose residue in the oligosaccharide linked to an Fc region of the antibody are reported in WO 1997/30087, Patel et al. See also document WO 1998/58964 (Raju, S.) and document WO 1999/22764 (Raju, S.) with reference to antibodies with altered carbohydrate linked to the Fc region of the same. See also US patent 2005/0123546 (Umana et al.) Which describes antibodies with modified glycosylation. Mutation of the consensus glycosylation sequence in the Fc region (Asn-X-Ser / Thr at positions 297 to 299, where X cannot be proline), for example, by mutating Asn of that sequence to any other amino acid, by placing a Pro at position 298, or by modifying position 299 to any amino acid except Ser or Thr, should counteract glycosylation at that position (see, for example, Fares Al-Ejeh et al., Clin. Cancer Res. (2007) 13: 5519s -5527s; Imperial! And Shannon, Biochemistry (1991) 30 (18): 4374-4380; Katsuri, Biochem J. (1997) 323 (Pt 2): 415-419; Shakin-Eshleman et al., J. Biol. Chem. (1996) 271: 6363-6366).
[00154] The term "hypervariable region", when used in the present application, refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (for example, residues 24 to 34 (L1), 50 to 56 (L2) and 89 to 97 (L3) in the variable domain of the light chain , and 31 to 35 (H1), 50 to 65 (H2) and 95 to 102 (H3) in the heavy chain variable domain (Kabat et al .. Sequences of Proteins of
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Immunological interest, 5- Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or these residues of a “hypervariable loop” (for example, residues 26 to 32 (L1), 50 to 52 (L2) and 91 to 96 (L3) in the variable domain of the light chain, and 26 to 32 (H1), 52A to 55 (H2) and 96 to 101 (H3) in the variable domain of the heavy chain (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). “FR” are those residues from the variable domain, except residues from the hypervariable region, as defined in the present application.
[001] A "full length antibody" is one that comprises an antigen binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The constant domains can be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.
[00155] A "naked antibody" is an antibody (as defined in the present application) that is not conjugated to a heterologous molecule, such as a cytotoxic component, polymer or radiolabel.
[00156] Antibody effector functions ”refer to those biological activities of an antibody that result in activation of the immune system, except activation of the complement pathway. These activities are widely found in the Fc region (a native sequence Fc region or a variant amino acid sequence Fc region) of an antibody. Examples of antibody effector functions include, for example, Fc receptor binding and antibody dependent cell mediated cytotoxicity (ADCC). In one embodiment, the antibody in the present application is essentially devoid of effector function. In another embodiment, the antibody in the present application maintains minimal effector function. Methods for modifying or eliminating effector function are well known in the art and include, but are not limited to
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92/171 a, eliminate all or a part of the Fc region responsible for the effector function (that is, with the use of an antibody or antibody fragment in a format devoid of all or a portion of the Fc region, as, but not limiting to, a Fab fragment, a single chain antibody, and the like, as described in the present application and known in the art; modifying the Fc region at one or more amino acid positions to eliminate the effector function (Fc binding impact: positions 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301,303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438 and 439; and modify the glycosylation of the antibody (including, but not limited to, producing the antibody in an environment that does not allow for wild-type mammalian glycosylation, remove one or more carbohydrate groups from an already glycosylated antibody, and modify the antibody at one or more amino acid positions acids to eliminate the ability of the antibody to be glycosylated at these positions (including, but not limited to, N297G and N297A).
[00157] Antibody "complement activation" functions, or properties of an antibody that allow or trigger "complement pathway activation" are used interchangeably, and refer to biological activities of an antibody that engages or stimulates the immune system's complement pathway in a subject. Such activities include, for example, Clq binding and complement-dependent cytotoxicity (CDC), and can be mediated by both the Fc and non-Fc portions of the antibody. Methods for modifying or eliminating the complement activation function are well known in the art and include, but are not limited to, eliminating all or a portion of the Fc region responsible for complement activation (that is, with the use of an antibody or fragment antibody in a format devoid of all or a portion of the Fc region,
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93/171 as, but not limited to, a Fab fragment, a single chain antibody, and the like as described in the present application and as is known in the art, or by modifying the Fc region at one or more amino acid positions to eliminate or mitigate interactions with complement components or the ability to activate complement components, such as positions 270, 322, 329 and 321 known to be involved in the C1q bond), and by modifying or eliminating a portion of the non-Fc region responsible for complement activation ( that is, modifying the CH1 region at position 132 (see, for example, Vidarte et al., (2001) J. Biol. Chem. 276 (41): 3821738223)).
[00158] Depending on the amino acid sequence of the constant domain of its heavy chains, full-length antibodies can be assigned to different "classes". There are five main classes of full-length antibodies: IgA, IgD, IgE, IgG and IgM, and several of these can further be divided into “subclasses” (isotypes), for example, lgG1, lgG2, lgG3, lgG4, IgA and lgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called alpha, delta, epsilon, gamma and mi, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
[00159] The term "recombinant antibody", as defined in the present application, refers to an antibody (e.g., chimeric, humanized, human antibody or antigen binding fragment thereof) that is expressed by a recombinant host cell that comprises nucleic acid encoding the antibody. Examples of “host cells” to produce recombinant antibodies include: (1) mammalian cells, for example, Chinese Hamster Ovary (CHO) cells, COS, myeloma cells (including Y0 and NS0 cells), kidney from hamster puppies
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94/171 (BHK), Hela and Vero cells; (2) insect cells, for example, sf9, sf21 and Tn5; (3) plant cells, for example, plants that belong to the genus Nicotians (for example, Nicotiana tabacurrí); (4) yeast cells, for example, those belonging to the genus Saccharomyces (for example, Saccharomyces cerevisiaé) or the Aspergillus genus (for example, Aspergillus nigery, (5) bacterial cells, for example, Escherichia coll cells or cells of Bacillus subtilis, etc.
[00159] As used in the present application, "which specifically binds" or "specifically binds" refers to an antibody that selectively or preferentially binds to an antigen. Binding affinity is usually determined using a standard assay, such as Scatchard analysis or surface plasmon resonance technique (for example, using BIACORE®).
[001] An "antibody that binds to the same epitope", such as a reference antibody, refers to an antibody that blocks the binding of the reference antibody to its antigen in a competitive test by 50% or more, and conversely, the reference antibody blocks the binding of the antibody to its antigen in a competition assay by 50% or more. In one embodiment, an anti-BACE1 antibody binds to the BACE1 epitope attached by YW412.8.31.
[002] The term "cytotoxic agent", as used in this application, refers to a substance that inhibits or prevents cell function and / or causes death or destruction of cells. Cytotoxic agents include, but are not limited to, radioactive isotopes (eg At ²¹¹ , l ¹³ⁱ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ^io3 , Bi ²¹² , P ³² , Pb ²¹² and radioactive isotopes of Lu ); chemotherapeutic agents or drugs, for example, methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other agents
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95/171 interleaving); growth inhibitory agents; enzymes and fragments thereof as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and the various antitumor or anticancer agents disclosed below.
[003] An "effective amount" of an agent, for example, a pharmaceutical formulation, refers to an effective amount in the dosages and for periods of time necessary to achieve the desired therapeutic or prophylactic result.
[004] The term "Fc region" in the present application is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, an Fc region of the human immunoglobulin heavy chain extends from Cys226 or Pro230 to the carboxyl termination of the heavy chain. However, Cterminal lysine (Lys447) from the Fc region may or may not be present. Unless otherwise specified in the present application, the numbering of amino acid residues in the Fc region or constant region is in accordance with the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological interest, 5- Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
[005] "Structure region" or "FR" refers to residues of variable domain, except residues of the hypervariable region (HVR). The RF of a variable domain generally consists of four FR domains: FR1, FR2, FR3 and FR4. Consequently, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1 (L1) -FR2-H2 (L2) -FR3-H3 (L3) ~ FR4.
[006] An “immunoconjugate” is an antibody conjugated to one or
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96/171 plus heterologous molecule (s) including, but not limited to, a marker or cytotoxic agent. Optionally, this conjugation is through a linker.
[007] A "linker" as used in the present application is a structure that covalently or non-covalently connects the anti-BBB-R antibody to the heterologous molecule. In certain embodiments, a linker is a peptide. In other embodiments, a binder is a chemical binder.
[008] A "marker" is a marker coupled with the antibody in the present application and used for detection or imaging. Examples of such markers include: radiomarkers, a fluorophore, a chromophore or an affinity marker. In one embodiment, the marker is a radio marker used for imaging, for example, tc99m or 1123, or a spin marker (spin lab} for nuclear magnetic resonance (NMR) imaging (also known as imaging by magnetic resonance, mri), like iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, iron, etc.
[009] An "individual" or subject "is a mammal. Mammals include, but are not limited to, domesticated animals (for example, cows, sheep, cats, dogs and horses), primates (for example, human and non-human primates such as monkeys), rabbits and rodents (for example, mice and rats) ). In certain embodiments, the individual or subject is a human.
[0010] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, an antibody is purified to more than 95% or 99% purity, as determined, for example, by electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillarity electrophoresis) or chromatographic methods (by ion exchange or reverse phase HPLC). For review of methods for assessing antibody purity, please consult
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97/171 example, Flatman etal., J. Chromatogr. B 848: 79-87 (2007).
[0011] The term “package insert” is used to refer to the instructions included as usual in commercial packaging of therapeutic products, which contain information on the indications, use, dosage, administration, combination therapy, against indications and / or warnings regarding the use of these therapeutic products.
[0012] The term "pharmaceutical formulation" refers to a preparation that is in such a way as to allow the biological activity of an active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to a subject to whom the formulation would be administered.
[0013] A pharmaceutically acceptable carrier ”refers to an ingredient in a pharmaceutical formulation, except an active ingredient, which is non-toxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer or preservative.
[0014] As used in the present application, "treatment" (and grammatical variations of the same as "treating" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed both for prophylaxis and during the course of clinical pathology. Desired treatment effects include, but are not limited to, preventing the occurrence or recurrence of the disease, relieving symptoms, decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, improving or relieving disease status and remission or prognosis for improvement. In some embodiments, the antibodies of the invention are used to slow the development of a disease or to slow the progression of a disease.
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IL Compositions and Methods
A. Production of Anti-BBB-R Antibodies and Conjugates of The Same [00160] The methods and articles of manufacture of the present invention use, or incorporate, an antibody that binds to a BBB-R. The BBB-R antigen to be used for the production or screening of antibodies can, for example, be a soluble form of a portion of it (e.g., the extracellular domain) of the BBB-R containing the desired epitope. Alternatively or in addition, cells that express BBB-R on their cell surfaces can be used to generate or select antibodies. Other forms and presentations of BBB-R useful for generating antibodies will be apparent to those skilled in the art. Examples of BBB-Rs in the present application include transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF-R), protein 1 related to the low density lipoprotein receptor (LRP1) and LRP8 etc. , glucose transporter 1 (Glutl) and growth factor similar to heparin-binding epidermal growth factor (HB-EGF).
[00161] In accordance with the present invention, a low-affinity anti-BBB-R antibody ”(for example, anti-TfR) is selected based on the data in the present application that demonstrate that these antibodies exhibit improved CNS absorption (by example, brain). In order to identify these low affinity antibodies, several assays for measuring antibody affinity are available, including, without limitation: Scatchard assay and surface plasmon resonance (for example, with the use of BIACORE®). According to an embodiment of the invention, the antibody has an affinity for the BBB-R antigen (for example, for TfR) of about 5 nM, or about 20 nM, or about 100 nM, about 50 μΜ, or about 30 μΜ, or about 10 μΜ, or about 1 μΜ, or about 500 nM. Thus, the affinity can be in the range of about 5 nM to about 50
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99/171 μΜ, or in the range of about 20 nM to about 30 μΜ, or in the range of about 30 nM to about 30 μΜ, or in the range of about 50 nM to about 1 μΜ, or in the range from about 100 nM to about 500 nM, for example, as measured by Scatchard or BIACORE® analysis In another embodiment of the invention, the antibody has a shorter dissociation half-life from the BBB-R antigen (for example, for TfR) than 1 minute, less than 2 minutes, less than 3 minutes, less than four minutes, less than 5 minutes, or less than 10 minutes to about 20 minutes, or up to about 30 minutes, as measured by competition link analysis BIACORE® [00162] Thus, the invention provides a method to produce an antibody useful for transporting a drug for neurological dysfunction through the blood-brain barrier, which comprises selecting an antibody from an antibody panel against a blood-brain barrier receptor (BBB-R ), because it has an affinity for the BBB-R which is in the range of about 5 nM, or about 20 nM, or about 100 nM, at about 50 μΜ, or about 30 μΜ, or about 10 μΜ, or about 1 μΜ, or about 500 mM. Thus, the affinity can be in the range of about 5 nM to about 50 μΜ, or in the range of about 20 nM to about 30 μΜ, or in the range of about 30 nM to about 30 μΜ, or in range of about 50 nM to about 1 μΜ, or in the range of about 100 nM to about 500 nM, for example, as measured by Scatchard or BIACORE® analysis. As understood by a person skilled in the art, the combination of a heterologous molecule / compound to an antibody, often decreases the affinity of the antibody to its target due, for example, to steric hindrance or even the elimination of a binding arm if the antibody is produced multispecific with one or more binding arms to a antigen different from the original target of the antibody. In one embodiment, a low affinity antibody of the invention, specific for BACE1-conjugated TfR had a Kd for TfR, as measured by
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BIACORE, of about 30 nM. In another embodiment, a low affinity antibody of the invention, specific for BACE1-conjugated TfR had a Kd for TfR, as measured by BIACORE, of about 600 nm. In another embodiment, a low-affinity antibody of the invention, specific for TfR conjugated to BACE1, had a Kd for TfR, as measured by BIACORE, of about 20 μΜ. In another embodiment, a low-affinity antibody of the invention, specific for TfR conjugated to BACE1, had a Kd for TfR, as measured by BIACORE, of about 30 μΜ.
[00163] An exemplary assay to assess antibody affinity is by Scatchard analysis. For example, the anti-BBB-R antibody of interest can be iodinated using the lactoperoxidase method (Bennett and Horuk, Methods in Enzymology 288 p.134-148 (1997)). A radiolabeled antiBBB-R antibody is purified from ¹²⁵ l-Na by gel filtration using a NAP-5 column and its specific activity is measured. 50 μι competition reaction mixtures. containing a fixed concentration of iodinated antibody and decreasing concentrations of unlabeled antibody serially diluted are plated in 96-well plates. Cells expressing BBB-R temporarily are grown in culture medium consisting of Eagle medium modified by Dulbecco (DMEM) (Genentech) supplemented with 10% FBS, L-glutamine 2 mM, penicillin-streptomycin , 1 mM at 37 ° C in CO 5% _2. The cells are separated from the dishes using Sigma Cell Dissociation Solution and washed with binding buffer (DMEM with 1% bovine serum albumin, 50 mM HEPES, pH 7.2 and 0.2% sodium azide). The washed cells are added to a density of approximately 200,000 cells in 0.2 ml of binding buffer to the 96-well plates containing 50 µl of the competition reaction mixture. The final concentration of the unlabeled antibody in the competition reaction with cells is varied, starting from 1000 nM and then decreasing to dilutions 1: 2 times to 10 concentrations and including a zero
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101/171 added, sample only buffer. Competition reactions with cells for each concentration of unlabeled antibody are evaluated in triplicate. Competition reactions with cells are incubated for 2 hours at room temperature. After 2 hours of incubation, the competition reactions are transferred to a filter plate and washed four times with binding buffer to separate the free antibody from the bound iodinated antibody. The filters are counted by the gamma counter and the binding data is evaluated using the adjustment algorithm of Munson and Rodbard (1980) to determine the binding affinity of the antibody.
[00164] An exemplary Scatchard analysis using the compositions of the invention can be performed as follows. Anti-TFR ^A was iodized using the lactoperoxidase method (Bennett and Horuk, Methods in Enzymology 288 p.134-148 (1997)). Anti-TFR ^A radiolabeled was purified from ^12b l-Na by gel filtration, using a NAP-5 column; ^the purified antiTFR had a specific activity of 19.82 pCi / pg. 50 µl competition reaction mixtures containing a fixed concentration of iodinated antibody and decreasing concentrations of unlabeled antibody serially diluted were plated in 96-well plates. 293 cells expressing murine TfR temporarily were cultured in culture medium consisting of Dulbecco's modified Eagle's medium (DMEM) (Genentech) supplemented with 10% FBS, 2 mM L-glutamine and 1 mM penicillin-streptomycin at 37 ^S C in CO 5% _2. The cells were separated from the dishes using Sigma Cell Dissociation Solution and washed with binding buffer (DMEM with 1% bovine serum albumin, 50 mM HEPES, pH 7.2 and 0.2% sodium azide). The washed cells were added to a density of approximately 200,000 cells in 0.2 ml of binding buffer to the 96-well plates containing 50 µl of the competition reaction mixture. The final concentration of iodinated antibody in each competition reaction with cells was 100 pM
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102/171 (134,000 cpm per 0.25 ml). The final concentration of the unlabeled antibody in the competition reaction with cells varied, starting from 1000 nM and then decreasing to dilutions 1: 2 times to 10 concentrations and including an added zero, sample only buffer. Competition reactions with cells for each concentration of unlabeled antibody were evaluated in triplicate. Competition reactions with cells were incubated for 2 hours at room temperature. After 2 hours of incubation, the competition reactions were transferred to a Millipore Multiscreen filter plate and washed four times with binding buffer to separate the free antibody from the bound iodinated antibody. The filters were counted in a Wallac Wizard 1470 gamma counter (PerkinElmer Life and Analytical Sciences; Waltham, MA). The binding data were evaluated using a New Ligand computer program (Genentech), which uses the adjustment algorithm of Munson and Rodbard (1980) to determine the binding affinity of the antibody.
[00165] An exemplary BIACORE® analysis using the compositions of the invention can be performed as follows. Kd was measured using surface plasmon resonance assays using BIACORE®-2000 (BIAcore, Inc., Piscataway, NJ) at 25 ⁹ C, using an anti-human Fc kit (BIAcore Inc., Piscataway, NJ). Briefly, carboxymethylated dextran (CM5, BIACORE Inc.) biosensor chips were activated with (3-dimethylaminopropyl) -carbodiimide (EDC) and N-hydroxysuccinimide (NHS) / V-ethyl-A / 'chloride, according to the supplier's instructions . The anti-human Fc antibody was diluted with 10 mM sodium acetate, pH 4.0, at 5 pg / mL prior to injection at a flow rate of 50 μΙ / minute to achieve approximately 10,000 response units (RU) of protein coupled. After antibody injection, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, monospecific or multispecific variant anti-TfR antibodies were injected into HBS-P to achieve
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103/171 about 220 RU, then two-fold serial dilutions of MuTfR-His (0.61 nM to 157 nM) were injected into HBS-P at 25 ^S C with a flow rate of approximately 30 μΙ / min. The association rates (kon) and dissociation rates (koff) were calculated using a Langmuir one-to-one connection model (BIACORE® Evaluation Computer Program version 3.2) while simultaneously adjusting the association and dissociation sensorgrams. The dissociation equilibrium constant (Kd) was calculated as the koff / kon ratio. See, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999) [00166] According to another embodiment, Kd is measured using plasmon resonance assays surface with Biacore®-2000 apparatus (BIAcore, Inc., Piscataway, NJ) at 25 ^to C, using an anti-human Fc kit (BIAcore Inc., Piscataway, NJ). Briefly, carboxymethylated dextran (CM5, BIACORE Inc.) biosensor chips are activated with N-ethyl-N ¹ - (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and / V-hydroxysuccinimide (NHS), according to the instructions of the provider. The anti-human Fc antibody is diluted with 10 mM sodium acetate, pH 4.0, at 5 pg / mL prior to injection at a flow rate of 50 μΙ / minute to achieve approximately 10,000 response units (RU) of protein coupled. After antibody injection, 1M ethanolamine is injected to block unreacted groups. For kinetic measurements, variant anti-BBBR-R antibodies are injected into HBS-P to achieve about 220 RU, then two-fold serial dilutions of BBB-R-His (0.61 nM to 157 nM) are injected into HBS -P to 25 ^to C with a flow rate of approximately 30 pl / min. The association rates (kon) and dissociation rates (koff) are calculated using a Langmuir one-to-one connection model (BIACORE® Evaluation Computer Program version 3.2) while simultaneously adjusting the association and dissociation sensorgrams. The dissociation equilibrium constant (Kd) is
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104/171 calculated as the koff / kon ratio. See, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999).
[00167] A surrogate measurement for the affinity of one or more antibodies to BBB-R is half of its maximum inhibitory concentration (IC50), a measure of how much of the antibody is needed to inhibit the binding of a known BBB-R ligand to BBB-R by 50%. Various methods for determining the IC 50 for a given compound are known in the art; a common approach is to perform a competition binding assay, as described in the present application in the examples, that is, in relation to Figure 1A. In general, a high IC50 indicates that something else from the antibody is needed to inhibit binding of the known ligand, and thus the antibody's affinity for the ligand is relatively low. Conversely, a low IC50 indicates that something less than the antibody is needed to inhibit binding of the known ligand, and thus the antibody's affinity for the ligand is relatively high.
[00168] An exemplary competitive ELISA assay for measuring IC50 is one in which the increased concentrations of anti-TfR or anti-TfR / brain antigen (i.e., anti-TfR / BACE1, anti-TfR / Abeta and the like) of antibodies variants are used to compete against biotinylated TfR ^A for binding to TfR. The anti-TfR competition ELISA was performed on Maxisorp plates (Neptune, NJ) coated with 2.5 pg / ml extracellular murine TfR domain purified in PBS at 4 ^S C overnight. The plates were washed with 0.05% PBS / Tween 20 and blocked using Superblock blocking buffer in PBS (Thermo Scientific, Hudson, NH). The titration of each individual anti-TfR or anti-TfR / brain antigen (ie, TÍR / BACE1 or antiTfR / Abeta) (1: 3 serial dilution) was combined with biotinylated anti-TfR ^A (final concentration 0.5 nM) and added to the plate for 1 hour at room temperature. The plates were washed with 0.05% PBS / Tween 20 and added
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HRP-streptavidin (Southern Biotech, Birmingham) to the plate and incubated for 1 hour at room temperature. The plates were washed with 0.05% PBS / Tween 20 and anti-TfR ^A biotinylate bound to the plate was detected using TMB substrate (BioFX Laboratories, Owings Mills).
[00169] In one embodiment, the low affinity anti-BBB-R antibody in the present application is coupled to a neurological dysfunction marker and / or drug or imaging agent in order to more efficiently transport the marker and / or drug or imaging agent through BBB. This coupling can be achieved by cross-linking chemicals or by generating fusion proteins, etc.
[00170] Covalent conjugation can be either direct or through a linker. In certain embodiments, direct conjugation is by constructing a fusion protein (that is, by genetic fusion of the two genes encoding the BBB-R antibody and the neurological dysfunction and expression drug as a single protein). In certain embodiments, direct conjugation is by forming a covalent bond between a reactive group in one of the two portions of the anti-BBB-R antibody and a corresponding or accepting group in the neurological drug. In certain embodiments, direct conjugation is by modification (ie, genetic modification) of one of the two molecules that will be conjugated to include a reactive group (such as non-limiting examples, a sulfhydryl group or a carboxyl group) that forms a covalent bond in another molecule that will be conjugated under appropriate conditions. As a non-limiting example, a molecule (i.e., an amino acid) with a desired reactive group (i.e., a cysteine residue) can be introduced, for example, into the anti-BBB-R antibody and a disulfide bond formed in the drug neurological. Methods for covalent conjugation of nucleic acids to proteins are also known in the art (ie, photoreticulation, see, for example, Zatsepin et al. Russ. Chem. Rev. 74: 77-95 (2005)). The conjugation
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106/171 non-covalent can be any means of non-covalent fixation, including hydrophobic bonds, ionic bonds, electrostatic interactions, and the like, as will be easily understood by a person skilled in the art. Conjugation can also be performed using a variety of binders. For example, an anti-BBB-R antibody and a neurological drug can be conjugated using a variety of bifunctional protein coupling agents such as N-succinimidyl-S-iS-pyridyldithium propionate (SPDP), succinimidil-4 - (Nmaleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), derivatives of bifunctional imidoesters (such as dimethyl adipimidate HCI), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis- (p -azidobenzoyl) hexanediamine), bis-diazonium derivatives (like bis- (p-diazoniumbenzoyl) ethylenediamine), diisocyanates (like toluene 2,6-diisocyanate) and bis-active fluorine compounds (like 1,5-difluoro-2, 4-dinitrobenzene). Peptide linkers can also be used, comprised of one to twenty amino acids joined by peptide bridges. In certain embodiments, amino acids are selected from the twenty naturally occurring amino acids. In certain embodiments, one or more of the amino acids are selected from glycine, alanine, proline, asparagine, glutamine and lysine. The ligand can be a "cleavable ligand" that facilitates the release of the neurological drug through distribution to the brain. For example, an acid-labile ligand, peptidase-sensitive ligand, photolabile ligand, dimethyl ligand or bisulfide-containing ligand (Chari et al., Cancer Res. 52: 127-131 (1992); US patent 5,208,020) can be used.
[00171] The invention in the present application expressly contemplates, but is not limited to those conjugates prepared with crosslinking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB,
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SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfoSMCC, and sulfo-SMPB, and SVSB (succinimidyl- (4-vinylsulfone) benzoate) that are commercially available (for example, together to Pierce Biotechnology, Inc., Rockford, IL., USA).
[00172] For a neuropathy dysfunction, a neurological drug can be selected, which is a pain reliever including, but not limited to, a narcotic / opioid pain reliever (ie, morphine, fentanyl, hydrocodone, meperidine, methadone, oxymorphone, pentazocine , propoxyphene, tramadol, codeine and oxycodone), a non-steroidal anti-inflammatory drug (NSAID) (i.e. ibuprofen, naproxen, diclofenac, diflunisal, etodolac, fenoprofen flurbiprofen, indomethacin, ketorolac, mefenamic acid, meloxicone, piroxicam, sulindac and tolmentin), a corticosteroid (ie, cortisone, prednisone and prednisolone, dexamethasone, methylprednisolone and triamcinolone), an anti-migraine agent (ie, sumatriptan, almotriptan, frovatriptan, sumatriptan, rizatriptan, zytripyriptane, zytripyriptane, zytripyriptane, zytripyriptan, zytripyriptan, zytripyroline, zytripyroline, zytripyroline, zytriptan, zeppyrine, zygyripyroline, , eletriptan and ergotamine), acetaminophen, a salicylate (ie aspirin, choline salicylate, magnesium salicylate, diflunisal and salsalate), an anticonvulsant (ie carb amazepine, clonazepam, gabapentin, lamotrigine, pregabalin, tiagabine and topiramate), an anesthetic (i.e., isoflurane, trichlorethylene, halothane, sevoflurane, benzocaine, chloroprocaine, cocaine, cyclometheine, dimetocaine, propoxicaine, procaine, procaine, nov , bupivacaine, carticaine, cinchocaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, piperocaine, prilocaine, ropivacaine, trimecain, saxitoxin and tetrodotoxin) and the cox-2 inhibitor (ie, celecoxib, rofecoxib) and vald. For a neuropathy disorder with vertigo involvement, a neurological drug that can be selected is an anti-vertigo agent including, but not limited to, meclizine, diphenhydramine, promethazine and diazepam. For a dysfunction of
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108/171 neuropathy with nausea involvement, a neurological drug that can be selected is an anti-nausea agent including, but not limited to, promethazine, chlorpromazine, prochlorperazine, trimetobenzamide and metoclopramide. For a neurodegenerative disease, a neurological drug that can be selected is a growth hormone or neurotrophic factor; examples include, but are not limited to, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophins-4/5, fibroblast growth factor-2 (FGF) and other FGFs, neurotrophin-3 (NT), erythropoietin (EPO), hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor-alpha (TGF), TGF-beta, vascular endothelial growth factor (VEGF), antagonist interleukin-1 receptor (IL-1ra), ciliary neurotrophic factor (CNTF), glial-derived neurotrophic factor (GDNF), neurturin, platelet-derived growth factor (PDGF), heregulin, neuregulin, artemine, persephin, interleukins, neurotrophic factor derived from glial cell lineage (GFR), granulocyte colony stimulating factor (SCF), macrophage and granulocyte CSF, netrins, cardiotrophin-1, hedgehogs, leukemia inhibitory factor (LIF), midkine, pleiotrophin, bone morphogenetic proteins (BMPs), netrins, saposins, semafori nas and stem cell factor (SCF).
[00173] For cancer, a neurological drug that is a chemotherapeutic agent can be selected. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; sulfonated alkyls such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide and trimethylolomelamine;
acetogenins (especially bulatacin and bulatacinone); delta-9tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapacone; lapacol;
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109/171 colchicines; betulinic acid; a camptothecin (including synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); briostatin; calistatin; CC-1065 (including its analogues adozelesin, carzelesin and synthetic byzelesin); podophyllotoxin; podophyllinic acid; teniposide; cryptoficina (particularly cryptoficina 1 and criptoficina 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleuterobin; pancratistatin; a sarcodictine; spongistatin; nitrogen mustards such as chlorambucil, chlornafazine, chlorophosphamide, estramustine, ifosfamide, meclorethamine, meclorethamide hydrochloride, melphalan, novembiquin, phenesterin, prednimustine, trophosphamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, photemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enedin antibiotics (eg calicheamicin, especially calicheamicin gamma 11 and calicheamicin omega 11 (see, for example, Agnew, Chem Intl. Ed. EngL, 33: 183-186 (1994)); dinemicin, including dinemicin A; a speramycin; as well as neocarzinostatin chromophores and enedin antiobiotic chromophores (related chromoproteins), aclacinomysins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophylline, chromomycin, diaromycin, dromomycin, dromomycin, dromomycin, dromomycin, dromomycin, diazine -L · norleukin, doxorubicin ADRIAMYCIN® (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin and deoxidoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, potomycin, mycomycin, potentiomycin, mycomycin, potentiomycin, mycomycin, potentiomycin, mycomycin, potentiomycin, mycomycin , puromycin, chelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues like
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110/171 fludarabine, 6-mercaptopurine, tiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, didesoxyuridine, doxifluridine, enocytabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutetimide, mitotane, trilostane; restorative folic acid like frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; ansacrine; bestrabucila; bisanthrene; edatraxate; defofamine; demecolcine; diaziquone; elfornitine; ellipinium acetate; an epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; fenamet; pirarubicin; losoxantrone; 2-ethyl hydrazide; procarbazine; PSK® polysaccharide complexes (JHS Natural Products, Eugene, OR); razoxan; rhizoxin; sizofirana; spirogermanio; tenuazonic acid; triaziquone; 2.2 ’, 2” -trichloro triethyl amine; trichothecenes (especially T2 toxin, verracurin A, roridin A and anguidine); urethane; vindesina (ELDISINE®, FILDESIN®); dacarbazine; manomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, for example, TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), Cremofor-free ABRAXANE ™, elaborated albumin nanoparticle paclitaxel formulation (American Pharmaceutical Partners, Schaumberg, Illinois) and TAXOTERE® doxetaxel (Rhône- Poulenc Rorer, Antony, France); chloranbucyl; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovine; vinorelbine (NAVELBINE®); new chair; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase RFS 2000 inhibitor; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); salts
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111/171 pharmaceutically acceptable acids or derivatives of any of the above; as well as combinations of two or more of the above like CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone, and FOLFOX, an abbreviation for an oxaliplatin (ELOXATIN ™) treatment regimen combined with 5-FU and leukovorin .
[00174] Also included in this definition of chemotherapeutic agents are anti-hormonal agents that act to regulate, reduce, block or inhibit the effects of hormones that can promote cancer growth, and are often in the form of systemic or body treatment. all. They can be the hormones themselves. Examples include antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen NOLVADEX®), raloxifene EVISTA®, droloxifene, 4-hydroxy tamoxifen, trloxlphen, queoxifene, LY117018, onapristone and toremif®; antiprogesterones; estrogen receptor infraregulators (ERDs); ovarian suppressing or blocking function agents, for example, hormone agonists that release luteinizing hormone (LHRH) such as LUPRON® and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetate and tripterelin; other antiandrogens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the aromatase enzyme, which regulates the production of estrogen in the adrenal glands such as, for example, 4 (5) -imidazoles, aminoglutetimide, megestrol acetate MEGASE®, exemestane AROMASIN®, formestane, fadrozola, vorozola RIVISOR®, letrozola FEMARA® and anastrozola ARIMIDEX®. In addition, this definition of chemotherapeutic agents includes bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid / zoledronate, FOSAMAX®alendronate, AREDIA® pamidronate,
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SKELID® tiludronate or ACTONEL® risedronate; as well as troxacitabine (a del del, 3-dioxolane cytosine nucleoside); antisense oligonucleotides, particularly those that inhibit the expression of genes in the signaling pathways involved in abnormal cell proliferation such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccine, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; topoisomerase 1 inhibitor LURTOTECAN% rmRH ABARELIX®; Apatinib iditosylate (a small molecule tyrosine kinase double inhibitor of ErbB-2 and EGFR also known as GW572016); and pharmaceutically acceptable salts, acids or derivatives of any of the above.
[00175] Another group of compounds that can be selected as neurological dredgers for cancer treatment or prevention are anti-cancer immunoglobulins (including, but not limited to, trastuzumab, pertuzumab, bevacizumab, alentuxumab, cetuximab, gemtuzumab ozogamycin, ibritumomabe tiuxetan, panitumumabe and rituitumumab and rituitumbebe ). In some cases, antibodies in conjunction with a toxic marker or conjugate can be used to target and destroy desired cells (i.e., cancer cells), including, but not limited to, tositumomab with a ¹³ Ί radiolabel or trastuzumab entansine.
[00176] For an eye disease or dysfunction, a neurological drug that can be selected is an antiangiogenic ophthalmic agent (ie, bevacizumab, ranibizumab and pegaptanib), a glaucoma ophthalmic agent (ie, carbacoi, epinephrine, demecarium bromide, apraclonidine, brimonidine, brinzolamide, levobunolol, timolol, betaxolol, dorzolamide, bimatoprost, carteolol, metipranolol, dipivephrine, travoprost and latanoprost), a carbonic anhydrase inhibitor (i.e. and tetrahydrozoline), a
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113/171 ocular lubricant, an ophthalmic steroid (i.e., fluorometholone, prednisolone, loteprednol, dexamethasone difluprednate, rimexolone, fluocinolone, medrisone and triamcinolone), an ophthalmic anesthetic (ie, lidocaine, proparacaine and an anti-tetanic acid and tetraacin) (ie, levofloxacin, gatifloxacin, ciprofloxacin, ofloxacin, chloramphenicol, bacitracin / polymyxin b, sulfacetamide, tobramycin, azithromycin, besifloxacin, norfloxacin, sulfisoxazole, gentamicin, oxyxinidine, tridoxycin, gyroxinidine, trichloridine, gyroxinidine, gyroxinidine, gyroxinidine, ), an ophthalmic anti-inflammatory agent (i.e., nepafenac, ketorolac flurbiprofen, suprofen, cyclosporine, triamcinolone, diclofenac and bronfenac), and an ophthalmic antihistamine or decongestant (ie, ketotifen, olopatadine, kinetin, epinastine, epinastine, epinastine, epinastamine tetrahydrozoline, pemirolast, bepotastine, naphazoline, phenylephrine, nedocromil, lodoxamide, phenylephrine, emedastine and azelastine).
[00177] For an epilepsy, a neurological drug that can be selected is an anticonvulsant or antiepletic including, but not limited to, barbiturate anticonvulsants (ie, primidone, metarbital, mefobarbital, alobarbital, amobarbital, aprobarbital, alfenal, barbital, bralobarbital and phenobarbital), benzodiazepine anticonvulsants (ie diazepam, clonazepam and lorazepam), carbamate anticonvulsants (ie, felbamate), carbonic anhydrase inhibiting anticonvulsants (ie, acetazolamide, topiramate and zonisamide) carbamazepine and oxcarbazepine), fatty acid derivatives (ie, divalproex and valproic acid), gamma-aminobutyric acid analogs (ie, pregabalin, gabapentin and vigabatrin), gamma-aminobutyric acid reuptake inhibitors (ie, tiagabine) gamma-aminobutyric acid inhibitors transaminase (ie vigabatrin), hydantoin anticonvulsant (ie phenytoin, etotoin, phosphenytoin and mephenytoin), various anticonvulsants (ie, lacosamide and magnesium sulfate),
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114/171 progestins (ie progesterone), anticonvulsant oxazolidinedione (ie, paramethadione and trimethadione), anti-convulsants pyrrolidine (ie, levetiracetam), succinimide (ie, etosuximide and methsuximide) anti-convulsants (ie, triazine) , and urea anti-convulsants (ie, phenacemide and feneturide).
[00178] For a lysosomal storage disease, a neurological drug that is itself or otherwise mimics the activity of the enzyme that is impaired by the disease can be selected. Exemplary recombinant enzymes for the treatment of lysosomal storage disorders include, but are not limited to, those presented, for example, in US patent application publication 2005/0142141 (i.e., alpha-L-iduronidase, iduronate-2-sulfatase, N -sulfatase, alpha-N-acetylglycosaminidase, N-acetyl-galactosamine-6-sulfatase, beta-galactosidase, arylsulfatase B, beta-glucuronidase, alpha-glucosidase acid, glycocerebrosidase, alpha-galactosidase A, hexosaminidase A, sphingomyelinase acid galactocerebrosidase, beta-galactosidase, arylsulfatase A, ceramidase acid, aspartoacylase, palmitoyl-protein thioesterase 1 and tripeptidyl amino peptidase 1).
[00179] For amyloidosis, a neurological drug that can be selected includes, but is not limited to, an antibody or other binding molecule (including, but not limited to, a small molecule, a peptide, an aptamer, or another protein ligand) that specifically binds to a target selected from: beta secretase, tau, presenilin, amyloid precursor protein or portions thereof, amyloid beta peptide, oligomers or fibrils thereof, death receptor 6 (DR6), rodent receptor advanced glycation endings (RAGE), parkin and huntingtin; a cholinesterase inhibitor (i.e., galantamine, donepezil, rivastigmine and tacrine); an NMDA receptor antagonist (i.e., memantine), a monoamine deplector (i.e., tetrabenazine); an ergoloid mesylate; an antiparkinsonism agent
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Anticholinergic (i.e., procyclidine, diphenhydramine, triexylphenidyl, benztropine, biperiden and triexiphenidyl); a dopaminergic antiparkinsonism agent (i.e., entacapone, selegiline, pramipexole, bromocriptine, rotigotine, selegiline, ropinirola, rasagiline, apomorphine, carbidopa, levodopa, pergolide, tolcapone and amantadine); tetrabenazine; an anti-inflammatory (including, but not limited to, a non-steroidal anti-inflammatory drug (i.e., indomethicin and other compounds listed above), a hormone (i.e., estrogen, progesterone and leuprolide); a vitamin (ie , folate and nicotinamide); a dimebolin; a homotaurin (ie, 3-aminopropanesulfonic acid; 3APS); a serotonin receptor activity modulator (ie, xaliprodene); an interferon and a glucocorticoid.
[00180] For a viral or microbial disease, a neurological drug that can be selected includes, but is not limited to, an antiviral compound (including, but not limited to, an adamantane antiviral (ie rimantadine and amantadine), a antiviral interferon (ie, peginterferon alfa2b), a chemokine receptor antagonist (ie, maraviroc), an integrase tape transfer inhibitor (ie, raltegravir), a neuraminidase inhibitor (ie, oseltamivir and zanamivir) , a non-nucleoside reverse transcriptase inhibitor (i.e., efavirenz, etravirine, delavirdine and nevirapine), a nucleoside reverse transcriptase inhibitor (tenofovir, abacavir, lamivudine, zidovudine, stavudine, entecavir, emtricitabine, telebivine, adefovir) , a protease inhibitor (i.e., darunavir, atazanavir, fosamprenavir, tipranavir, ritonavir, nelfinavir, amprenavir, indinavir, saquinavir), a nucleoside purine (i.e., valacyclovir, fanciclovir, acyclovi r, ribavirin, ganciclovir, valganciclovir and cidofovir), and a varied antiviral (i.e., enfuvirtide, foscarnet, palivizumab and fomivirsen)), an antibiotic (including, but not limited to, an aminopenicillin (ie, amoxicillin, ampicillin, chloramphenicol, nafcillin, cloxacillin, dicloxacillin, flucoxacillin, temocillin,
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116/171 azlocillin, carbenicillin, ticarcillin, mezlocillin, piperacillin and bacampicillin), a cephalosporin (i.e., cefazolin, cephalexin, cephalotin, cefamandola, ceftriaxone, cefotaxime, ceftazine, ceftazefe, cefacefe, cefa, cefoxitin), a carbapenem / penem (ie imipenem, meropenem, ertapenem, faropenem and doripenem), a monobactam (ie aztreonam, tigemonam, norcardicin A and tabtoxinin-beta-lactam, a beta-lactam inhibitor (ie , clavulanic acid, tazobactam and sulbactam), in conjunction with another beta-lactam antibiotic, an aminoglycoside (i.e., amikacin, gentamicin, kanamycin, neomycin, netylmycin, streptomycin, tobramycin and paromomycin), an ansamycin (ie geldanamycin and herbimycin ), a carbacefem (ie, loracarbef), a glycopeptide (ie, teicoplanin and vancomycin), a macrolide (ie, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin and spectinomycin), a monobactam (ie, aztreonam), a quinolone (ie, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, esofpaoxoxin, isofloxacin, grepafloxin, and , mafenide, sulfonamidocrisoidine, sulfacetamide, sulfadiazine, sulfamethizole, sulfanilamide, sulfasalazine, sulfisoxazole, sulfamethoxazole, trimethoprim and sulfamethoxazole), tetracycline (ie, tetracycline, demeclocycline, oxycycline, oxycycline, oxycycline, and , mitoxantrone, bleomycin, daunorubicin, dactinomycin, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin and valrubicin) and various antibacterial compounds (ie, bacitracin, colistin and polymyxin B)), an antifungal (ie, metronidazole, nitronidazole, chloroquine, iodoquinol and paromomycin), and an antiparasitic (including, but not limited to, quinine, chloroquine, amodiaquine, pyrimethamine, sulfadoxine,
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117/171 proguanil, mefloquine, atovaquone, primaquine, artemesinin, halofantrine, doxycycline, clindamycin, mebendazole, pyrantel pamoate, thiabendazole, diethylcarbamazine, ivermectin, rifampin, amphotericin B, melarsoprol, and efornine.
[00181] For ischemia, a neurological drug that can be selected includes, but is not limited to, a thrombolytic agent (ie, urokinase, alteplase, reteplase and tenecteplase), platelet aggregation inhibitor (ie, aspirin, cilostazol, clopidogrel , prasugrel and dipyridamole), a statin (i.e., lovastatin, pravastatin, fluvastatin, rosuvastatin, atorvastatin, simvastatin, cerivastatin and pitavastatin), and a compound to improve blood flow or vascular flexibility, including, for example, blood pressure medications .
[00182] For behavioral dysfunction, a neurological drug that can be selected from a compound that modifies behavior including, but not limited to, an atypical antipsychotic (i.e., risperidone, olanzapine, apripiprazole, quetiapine, paliperidone, asenapine , clozapine, iloperidone and ziprasidone), an antipsychotic phenothiazine (ie, prochlorperazine, chlorpromazine, fluphenazine, perfenazine, trifluoperazine, thioridazine and mesoridazine), a thioxanthene (ie, thiotixene), a pesticide, is a pesticide, molindone, haloperidol and loxapine), a selective serotonin reuptake inhibitor (i.e., citalopram, escitalopram, paroxetine, fluoxetine and sertraline), a serotoninanorepinephrine reuptake inhibitor (ie, duloxetine, venlafaxine, an antidiabetic agent , doxepin, clomipramine, amoxapine, nortriptyline, amitriptyline, trimipramine, imipramine, protriptyline and desipramine), a tetracyclic antidepressant ico (ie, mirtazapine and maprotiline), a phenylpiperazine antidepressant (ie, trazodone and nefazodone), a monoamine oxidase inhibitor (ie, isocarboxazide, phenelzine, selegiline and
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118/171 tanilcipromina), a benzodiazepine (i.e., alprazolam, estazolam, flurazeptam, clonazepam, lorazepam and diazepam), a noradrenaline-dopamine reuptake inhibitor (ie, bupropion), a CNS stimulant (ie, phentermine diethylpropion, methamphetamine, dextroamphetamine, amphetamine, methylphenidate, dexmethylphenidate, lysdexamphetamine, modafinil, pemoline, phendimetrazine, benzophetamine, phendimetrazine, armodafinil, diethylpropion, an anti-narcotic, and an antioxidant, butoxin, an antioxidant and an antioxidant; a, a barbiturate (i.e., secobarbital, phenobarbital and mefobarbital), a benzodiazepine (as described above), and a varied anxiolytic / sedative / hypnotic (ie, diphenhydramine, sodium oxybate, zaleplon, hydroxyzine, chloral hydrate, aolpidem , buspirone, doxepine, eszopiclone, ramelteon, meprobamate and etchlorvinol)), a secretin (see, for example, Ratliff-Schaub et al. Autism 9: 256-265 (2005)), an opioid peptide (see, for example, Cowen et a /., J. Neurochem. 89: 273-285 (2004)), and a neuropeptide (see, for example, Hethwa et al. Am. J. Physiol. 289: E301-305 (2005)).
[00183] For CNS inflammation, a neurological drug that targets the inflammation itself (ie, a non-steroidal anti-inflammatory such as ibuprofen or naproxen) or one that treats the underlying causes of the inflammation (ie, an antiviral agent) and anticancer).
[00184] According to an embodiment of the invention, "coupling" is obtained by generating a multispecific antibody (for example, a bispecific antibody). Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens or epitopes. In one embodiment, the multispecific antibody comprises a first antigen binding site that binds to BBB-R and a second antigen binding site that binds to the brain antigen, such as beta-secretase 1 (BACE1) or Abeta and the others
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119/171 brain antigens disclosed in the present application.
[00185] An exemplary brain antigen bound by the multispecific / bispecific antibody is BACE1, and an exemplary antibody that binds to it is the YW412.8.31 antibody in Figures 9A-B in the present application.
[00186] In another embodiment, the cerebral antigen is Abeta, exemplary as antibodies that are described in WO 2007068412, W02008011348, W020080156622 and W02008156621, expressly incorporated into the present application as a reference, with an exemplary Abeta antibody comprising MABT5102A lgG4 which comprises the amino acid sequences of the heavy and light chains in Figures 11A and 11B, respectively.
[00187] Techniques for making multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy-light chain pairs that have different specificities (see, Milstein and Cuello, Nature 305: 537 (1983)) , WO 93/08829, and Traunecker et at., EMBO J. 10: 3655 (1991)), and elaboration of "orifice bulge" (see, for example, US patent 5,731,168). Multispecific antibodies can also be produced by elaborating electrostatic targeting effects for antibody production of Fc heterodimeric molecules (WO 2009 / 089004A1); cross-linking of two or more antibodies or fragments (see, for example, US patent 4,676,980, and Brennan et al., Science, 229: 81 (1985)); with the use of leucine zippers to produce bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992)); with the use of diabody technology ”to produce bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 64446448 (1993)); and with the use of single-chain Fv (sFv) dimers (see, for example, Gruber et al., J. Immunol., 152: 5368 (1994)); and preparing antibodies
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120/171 triespecifics as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991).
[00188] Antibodies genetically engineered with three or more functional antigen binding sites, including "octopus antibodies" or "double variable domain immunoglobulins" (DVDs), are also included in this application (see, for example, US patent 2006 / 0025576A1 and Wu et al. Nature Biotechnology (2007)).
[00189] The antibody or fragment in the present application also includes a "Double Action FAb" or "DAF" comprising an antigen binding site that binds to BBB-R (e.g., TfR) as well as a brain antigen (e.g., BACE1) (see, US patent 2008/0069820, for example).
[00190] In one embodiment, the antibody is an antibody fragment, several of which are disclosed above. In another embodiment, the antibody is an intact or whole antibody. Depending on the amino acid sequence of the constant domain of its heavy chains, intact antibodies can be assigned to different classes. There are five main classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses (isotypes), for example, lgG1, lgG2, lgG3, lgG4, IgA and lgA2. 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. In one embodiment, the intact antibody is devoid of effector function. In one embodiment, the intact antibody has reduced effector function.
[00191] Techniques for generating antibodies and examples provided above are known in the definitions section of this document. In a
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121/171 embodiment, the antibody is a chimeric, humanized or human antibody, or antigen binding fragment thereof.
[00192] Several techniques are available for determining antibody binding to BBB-R. One such assay is an enzyme-linked immunosorbent assay (ELISA) to confirm the ability to bind to human BBB-R (and brain antigen). According to that assay, antigen-coated plates (for example, recombinant BBB-R) are incubated with a sample comprising the anti-BBB-R antibody and binding of the antibody to the antigen of interest is determined.
[00193] In one aspect, an antibody of the invention is tested for its antigen binding activity, for example, by methods known as ELISA, Western blot, etc.
[00194] Assays to assess absorption of systemically administered antibody and other biological activities of the antibody can be performed as disclosed in the examples or as is known for the anti-CNS antigen antibody of interest.
[00195] Exemplary assays where the multispecific antibody binds to BACE1 should now be described.
[00196] Competition assays can be used to identify an antibody that competes with any of the antiBACE1 antibodies or Fabs described in the present application, for example, YW412.8, YW412.8.31, YW412.8.30, YW412.8.2, YW412.8.29 , YW412.8.51, Fab12, LC6, LC9, LC10 for binding to BACE1. In certain embodiments, that competition antibody binds to the same epitope (for example, a linear or a conformational epitope) that is bound by any of the anti-BACE1 antibodies or Fabs described in the present application, for example, YW412.8, YW412 .8.31, YW412.8.30, YW412.8.2, YW412.8.29, YW412.8.51, Fab12, LC6, LC9, LC10. Detailed exemplary methods for mapping an epitope to which an antibody
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122/171 alloys are provided in Morris (1996) "Epitope Mapping Protocols", in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).
[00197] In an exemplary competition assay, immobilized BACE1 is incubated in a solution that comprises a first labeled antibody that binds to BACE1 (for example, YW412.8, YW412.8.31, YW412.8.30, YW412.8.2, YW412 .8.29, YW412.8.51, Fab12, LC6, LC9, LC10) and a second unlabeled antibody being tested for its ability to compete with the first antibody for BACE1 binding. The second antibody can be present in a hybridoma supernatant. As a control, immobilized BACE1 is incubated in a solution that comprises the first labeled antibody, but not the second unlabeled antibody. After incubation under permissive conditions for binding the first antibody to BACE1, excess unbound antibody is removed and the amount of marker associated with immobilized BACE1 is measured. If the amount of marker associated with immobilized BACE1 is substantially reduced in the test sample relative to the control sample, then this indicates that the second antibody is competing with the first antibody for binding to BACE1. See Harlow and Lane (1988) Antibodies: A Laboratory Manual, chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
[00198] In one aspect, assays are provided to identify anti-BACE1 antibodies from those that have biological activity. Biological activity can include, for example, inhibition of BACE1 aspartyl protease activity. Antibodies that have biological activity in vivo and / or in vitro are also provided, for example, as assessed by a homogeneous time resolved fluorescence assay HTRF or a microfluidic capillary electrophoretic assay (MCE) using synthetic substrate peptides , or in vivo in cell lines that express BACE1 substrates as APP.
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123/171 [00199] The antibody (including the multispecific antibody) in the present application is optionally produced recombinantly in a host cell transformed with nucleic acid sequences encoding its heavy and / or light chains (for example, where the host cell or cells hosts were transformed by one or more vectors with nucleic acid in it). The host cell (s) is (are) a mammalian cell, for example, a Chinese Hamster Ovary Cell (CHO).
B. Pharmaceutical Formulations [00200] Therapeutic formulations of antibodies used according to the present invention are prepared for storage by mixing the antibody having the desired purification degree with pharmaceutically acceptable excipients , or stabilizers optional vehicles (Remington's Pharmaceutical Sciences 16th edition , Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol) ; low molecular weight polypeptides (less than approximately 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers like polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars like sucrose, mannitol, trehalose or sorbitol; formation of counter ions salts such as sodium; metal complexes (for example, Zn-protein complex); and / or
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124/171 non-ionic surfactants such as TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG).
[00201] The formulation in the present application can also contain more than one active compound, optionally those with complementary activities that do not adversely affect each other. The type and effective amounts of such drugs depend, for example, on the amount of antibody present in the formulation, and the subjects' clinical parameters. Examples of these drugs are discussed below.
[00202] The active ingredients can also be retained in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly (methylmethacrylate) microcapsule, respectively, in distribution systems of colloidal drug (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. These techniques are disclosed, for example, in Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). One or more therapeutic agents can be encapsulated in liposomes that are coupled to anti-BBB-R (see, for example, US patent application publication 20020025313).
[00203] Sustained release preparations can be prepared. Suitable examples of continuous release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, whose matrices are in the form of modeled articles, for example, films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (for example, poly (2-hydroxyethylmethacrylate) or poly (vinyl alcohol), polylactides (US patent copolymers of L-glutamic acid and γ-ethyl-L-glutamate, ethylene vinyl acetate non-degradable, degradable lactic acid-glycolic acid copolymers like LUPRON
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DEPOT ™ (injectable microspheres composed of copolymers lactic acid, glycolic acid and leuprolide acetate), and poly-D - (-) - 3-hydroxybutyric acid.
[00204] The formulations to be used for in vivo administration must be sterile. This is easily accomplished by filtration, through sterile filtration membranes.
[00205] In one embodiment, the formulation is isotonic.
C. THERAPEUTIC USES OF ANTHBBB-R ANTIBODIES [00206] The anti-BBB-R antibodies (including multispecific antibodies that comprise them) of the invention can be used in a variety of in vivo methods. For example, the invention provides a method of transporting a therapeutic compound across the blood-brain barrier with reduced or eliminated impact on red blood cell populations, which comprises exposing the anti-BBB-R antibody coupled to a therapeutic compound (e.g. a multispecific antibody that binds to both BBB-R and a brain antigen) to BBB, so that the antibody carries the therapeutic compound coupled to it through the BBB. In another example, the invention provides a method of transporting a drug for neurological dysfunction through the blood-brain barrier, which comprises exposing an anti-BBB-R antibody of the invention coupled to a drug for brain dysfunction (for example, a multispecific antibody that it binds both BBB-R and a brain antigen) to BBB, so that the antibody carries the drug for neurological dysfunction coupled to it with reduced or eliminated impact on red blood cell populations. In one embodiment, the BBB of the present application is in a mammal (for example, a human), for example, one that has neurological dysfunction, including, without limitation: Alzheimer's disease (AD), stroke, dementia, dystrophy muscle (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS),
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126/171 cystic fibrosis, Angelman syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, traumatic brain injury, etc.
[00207] In one embodiment, neurological dysfunction is selected from: neuropathy, amyloidosis, cancer (for example, involving the CNS or brain), an eye disease or dysfunction, a viral or microbial infection, inflammation (for example, from CNS or brain), ischemia, neurodegenerative disease, seizure, behavioral disorder, lysosomal storage disease, etc.
[00208] Neuropathy dysfunctions are diseases or abnormalities of the nervous system characterized by inadequate or uncontrolled nerve signaling or lack thereof, and include, but are not limited to, chronic pain (including nociceptive pain) pain caused by an injury to body tissues, including cancer-related pain, neuropathic pain (pain caused by abnormalities in the nerves, spinal cord or brain) and psychogenic pain (completely or mainly related to a psychological dysfunction), headache, migraine, neuropathy and symptoms and syndromes who often accompany these neuropathy disorders such as vertigo or nausea.
[00209] Amyloidoses are a group of diseases and disorders associated with extracellular protein deposits in the CNS, which include, but are not limited to secondary amyloidosis, age-related amyloidosis, Alzheimer's disease (AD), mild cognitive impairment (MCI) , Lewy body dementia, Down syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type), Parkinson-Guam dementia complex, cerebral amyloid angiopathy, Huntington's disease, progressive supranuclear palsy, multiple sclerosis, Creutzfeld Jacob disease, Parkinson's, transmissible spongiform encephalopathy, dementia
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127/171 related to HIV, amyotrophic lateral sclerosis (ALS), myositis with inclusion body (IBM) and eye diseases related to beta-amyloid deposition (ie macular degeneration, druse and cataract-related optic neuropathy).
[00210] CNS cancers are characterized by abnormal proliferation of one or more CNS cells (ie, a neural cell) and include, but are not limited to, glioma, glioblastoma multiforme, meningioma, astrocytoma, acoustic neuroma, chondroma, oligodendroglioma , medulloblastoma, ganglioglioma, Schwannoma, neurofibroma, neuroblastoma and extradural, intramedullary or intradural tumors.
[00211] Eye diseases or disorders are diseases or disorders of the eye, which for the purposes of this application is considered an organ of the CNS segregated by the BBB. Eye disorders or dysfunctions include, but are not limited to, dysfunctions of the sclera, cornea, iris and ciliary body (ie, scleritis, keratitis, corneal ulcer, corneal abrasion, snow blindness, bow eye, dotted superficial keratopathy) Thygeson, corneal neovascularization, Fuchs dystrophy, keratoconus, sicca keratoconjunctivitis, iritis and uveitis), lens dysfunctions (ie, cataracts), choroidal and retinal disorders (i.e., retinal displacement, retinoschisis, diabetic hypertensive retinopathy, retinopathy , retinopathy, retinopathy of prematurity, age-related macular degeneration, macular degeneration (dry or wet), epiretinal membrane, pigmentous retinitis and macular edema), glaucoma, floaters, optic nerve dysfunction and visual pathways (ie, hereditary optic neuropathy Leber and optic disc druses), eye muscle diseases / accommodation and retraction of binocular movement (ie strabismus, ophthalmoparesis, progre external ophthalmoplegia vision, esotropia, exotropia, hyperopia, myopia, astigmatism, anisometropia, presbyopia and ophthalmoplegia), visual disturbances and blindness (ie amblyopia, amaurosis
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128/171 Lever's congenital, scotoma, color blindness, achromatopsia, nictalopia, blindness, river blindness and micro-ophthalmia / coloboma), red eyes, Argyll Robertson's pupil, keratomycosis, xerophthalmia and aniridia.
[00212] Viral or microbial infections of the CNS include, but are not limited to, virus infections (i.e., influenza, HIV, poliovirus, rubella), bacteria (i.e., Neisseria sp., Streptococcus sp., Pseudomonas sp., Proteus sp., E. coii, S. aureus, Pneumococcus sp., Meningococcus sp., Haemophilus sp. And Mycobacterium tuberculosis) and other microorganisms such as fungi (ie yeast, Cryptococcus neoformans), parasites (ie, Toxoplasma gondii) or amoebas that result in CNS pathophysiology, which include, but are not limited to, meningitis, encephalitis, myelitis, vasculitis and abscess, which can be acute or chronic.
[00213] Inflammation of the CNS, includes, but is not limited to, inflammation that is caused by an injury to the CNS, which may be a physical injury (ie, due to accidents, surgery, head trauma, spinal cord injury, concussion ) and an injury due to or related to one or more other diseases or dysfunctions of the CNS (ie, abscess, cancer, viral or microbial infection).
[00214] CNS ischemia, as used in the present application, refers to a group of disorders related to blood flow or aberrant vascular behavior in the brain or the causes for it, and include, but are not limited to: focal cerebral ischemia , global cerebral ischemia, stroke (ie, subarachnoid hemorrhage or intracerebral hemorrhage) and aneurysm.
[00215] Neurodegenerative diseases are a group of diseases and disorders associated with loss of function or death of neural cells in the CNS, and include, but are not limited to: adrenoleukodystrophy, Alexander disease, Alper's disease, amyotrophic lateral sclerosis, ataxia
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129/171 telangiectasia, Batten's disease, Cockayne syndrome, corticobasal degeneration, degeneration caused by or associated with an amyloidosis, Friedreich's ataxia, frontotemporal lobar degeneration, Kennedy's disease, multiple system atrophy, multiple sclerosis, primary lateral sclerosis, paralysis progressive supranuclear, spinal muscular atrophy, transverse myelitis, Refsum's disease and spinocerebellar ataxia.
[00216] CNS seizure disorders and disorders involve inappropriate and / or abnormal electrical conduction in the CNS, and include, but are not limited to, epilepsy (ie, absence seizures, atonic seizures, benign rolandic epilepsy, childhood absence, chronic seizures, complex partial seizures, frontal lobe epilepsy, febrile seizures, infantile spasms, juvenile myoclonic epilepsy, juvenile absence epilepsy, Lennox-Gastaut syndrome, Landau-Kleffner syndrome, Dravet syndrome, Otahara syndrome, West syndrome , myoclonic seizures, mitochondrial dysfunctions, progressive myoclonic epilepsy, psychogenic seizures, reflex epilepsy, Rasmussen syndrome, simple partial seizures, secondarily generalized seizures, temporal lobe epilepsy, tonic-clonic seizures, tonic seizures, psychomotor seizures, psychomotor seizures, psychomotor seizures, partial onset, generalized onset seizures, status epilepticus, abdominal epilepsy inhalation, akinetic seizures, autonomic seizures, massive bilateral myoclonus, catamenial epilepsy, seizure fall, emotional seizures, focal seizures, gelastic seizures, Jacksonian seizures, Lafora disease, motor seizures, multifocal seizures, nocturnal seizures, seizure seizures, seizure seizures sensory seizures, subtle seizures, Sylvan seizures, withdrawal seizures and visual reflex seizures).
[00217] Behavioral disorders are disorders of the
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CNS characterized by aberrant behavior on the part of the afflicted subject and include, but are not limited to, sleep disorders (ie, insomnia, parasonies, night terrors, dysfunction of the circadian rhythm of sleep and narcolepsy), mood disorders (ie, depression, suicidal depression, anxiety, affective chronic illnesses, phobias, panic attacks, obsessive compulsive disorder, attention deficit hyperactivity disorder (ADHD), attention deficit disorder (ADD), chronic fatigue syndrome, agoraphobia, post-traumatic stress disorder, bipolar disorder), eating disorders (ie, anorexia or bulimia), psychoses, developmental behavior disorders (ie, autism, Rett syndrome, Aspberger syndrome), personality disorders and psychotic disorders (ie ie, schizophrenia, delusional disorder and the like).
[00218] Lysosomal deposit disorders are metabolic disorders that are, in some cases, associated with the CNS or have specific symptoms in the CNS; these disorders include, but are not limited to: Tay-Sachs disease, Gaucher disease, Fabry disease, mucopolysaccharidosis (types I, II, III, IV, V, VI and VII), glycogen storage diseases, GM1- gangliosidosis, metachromatic leukodystrophy, Farber's disease, Canavan leukodystrophy and neuronal ceroid lipofuscinosis types 1 and 2, Niemann-Pick disease, Pompe disease and Krabbe disease.
[00219] In another embodiment, diseases related to or caused by inappropriate overproduction of red blood cells, or in which overproduction of red blood cells is an effect of the disease, can be prevented or treated by the depletion effect of reticulocytes recognized in the present application of anti antibodies -TfR that maintain at least a partial effector function. For example, in congenital or neoplastic polycythemia, high red blood cell counts due to hyperproliferation, for example, reticulocytes, result in blood thickening and symptoms
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131/171 concomitant physiological. Lab. Haematol. (1996) Suppl. 1: 29-34). Administration of an anti-TfR antibody of the invention in which at least the partial effector function of the antibody has been preserved would allow selective removal of immature reticulocytes without affecting normal transferrin transport in the CNS. The dosage of this antibody could be modulated, so that these acute clinical symptoms could be minimized (that is, by dosing a very low dose or at widely spaced intervals), as is well understood in the art.
[00220] In one aspect, an antibody of the invention is used to detect neurological dysfunction prior to the onset of symptoms and / or to assess the severity or duration of the disease or dysfunction. In one aspect, the antibody allows the detection and / or imaging of neurological dysfunction, including imaging by radiography, tomography or magnetic resonance imaging (MRI).
[00221] In one aspect, a low affinity anti-BBBR antibody of the invention is provided for use as a medicament. In additional aspects, a low affinity anti-BBB-R antibody is provided for use in the treatment of a neurological disease or dysfunction (e.g. Alzheimer's disease) without red blood cell depletion (i.e., reticulocytes). In certain embodiments, a modified low-affinity anti-BBB-R antibody for use in a treatment method is provided, as described in the present application. In certain embodiments, the invention provides an anti-BBB-R antibody modified to enhance its safety for use in a method of treating an individual who has a neurological disorder or disorder, which comprises administering to the individual an effective amount of the anti-BBB antibody -R (optionally coupled to a drug for neurological dysfunction). In that embodiment, the method further comprises administering to the individual an effective amount of at least one therapeutic agent
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Additional 132/171. In additional embodiments, the invention provides an antiBBB-R antibody modified to enhance its safety for use in reducing or inhibiting the formation of amyloid plaque in a patient at risk or suffering from a neurological disease or dysfunction (for example, Alzheimer's disease) . An "individual", according to any of the above achievements, is optionally a human. In certain aspects, the anti-BBB-R antibody of the invention for use in the methods of the invention improves the absorption of the drug for neurological dysfunction with which it is coupled.
[00222] In a further aspect, the invention provides the use of a low affinity anti-BBB-R antibody of the invention in the manufacture or preparation of a medicament. In one embodiment, the drug is for treating neurological disease or dysfunction. In a further embodiment, the drug is for use in a method of treating neurological disease or dysfunction which comprises administering to an individual who has neurological disease or dysfunction an effective amount of the drug. In that embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent.
[00223] In a further aspect, the invention provides a method for treating Alzheimer's disease. In one embodiment, the method comprises administering to an individual who has Alzheimer's disease, an effective amount of a multispecific antibody of the invention that binds both BACE1 and TfR, or both, Abeta and TfR. In that embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. An "individual", according to any of the above achievements, can be a human.
[00224] The anti-BBB-R antibodies of the invention can be used either alone or in combination with other agents in a therapy. For example, the anti-BBB-R antibody of the invention can be
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133/171 co-administered with at least one additional therapeutic agent. In certain embodiments, an additional therapeutic agent is an effective therapeutic agent to treat oneself or a different neurological dysfunction, such as the anti-BBB-R antibody being employed to treat. Exemplary additional therapeutic agents include, but are not limited to: various neurological drugs described above, cholinesterase inhibitors such as donepezil, galantamine, rovastigmine and tacrine), NMDA receptor antagonists (such as memantine), amyloid beta peptide aggregation inhibitors, antioxidants, γ-secretase modulators, NGF gene therapy or mimic nerve growth factor (NGF) mimics, PPARy agonists, HMS-CoA reductase inhibitors (statins), ampakines, calcium channel blockers, GABA receptor antagonists, inhibitors glycogen synthase kinase, intravenous immunoglobulin, muscarinic receptor agonists, nicrotinic receptor modulator, active or passive beta amyloid peptide immunization, phosphodiesterase inhibitors, serotonin receptor antagonists and anti-amyloid beta peptide antibodies. In certain embodiments, at least one additional therapeutic agent is selected for its ability to mitigate one or more side effects of the neurological drug.
[00225] As exemplified in the present application, certain anti-BBB-R antibodies can have side effects that negatively impact reticulocyte populations in a subject treated with the anti-BBB-R antibody. Thus, in certain embodiments, at least one additional therapeutic agent selected for its ability to mitigate these negative side effects in reticulocyte populations is co-administered with an anti-BBB-R antibody of the invention. Examples of such therapeutic agents include, but are not limited to, agents to increase populations of red blood cells (i.e., reticulocytes), agents to support the growth and development of red blood cells (i.e., reticulocytes), and
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134/171 agents to protect red blood cell populations from the effects of the anti-BBB-R antibody; these agents include, but are not limited to, erythropoietin (EPO), iron supplements, vitamin C, folic acid and vitamin B12, as well as physical replacement of red blood cells (ie reticulocytes), for example, by transfusion of similar cells that may be from another individual of similar blood type or may have been previously extracted from the subject to which the anti-BBB-R antibody is administered. It will be understood by a person skilled in the art that, in some cases, existing agents designed to protect red blood cells (i.e. reticulocytes) are preferably administered to the subject, before or simultaneously with anti-BBB-R antibody therapy, while the agents designed to support or initiate renewed growth / development of red blood cells or populations of red blood cells (i.e., reticulocytes or populations of reticulocytes) are preferably administered simultaneously with or after anti-BBB-R antibody therapy, so that red blood cells can be replaced after treatment with anti-BBB-R antibody.
[00226] In other embodiments, at least one additional therapeutic agent is selected for its ability to inhibit or prevent activation of the complement pathway by administering the anti-BBB-R antibody. Examples of such therapeutic agents include, but are not limited to, agents that interfere with the ability of the anti-BBBR antibody to bind or activate the complement pathway and agents that inhibit one or more molecular interactions in the complement pathway, and are generally described in Mollnes and Kirschfink (2006) Molec. Immunol. 43: 107-121, the contents of which are expressly incorporated into this application as a reference.
[00227] These therapy combinations noted above encompass combined administration (when two or more therapeutic agents are included in the same formulation or separate), and
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135/171 separate administration, in that case, administration of the antibody of the invention can occur before, simultaneously and / or after administration of the additional therapeutic agent and / or adjuvant. Antibodies of the invention can also be used in combination with other intervention therapies such as, but not limited to, radiation therapy, behavioral therapy or other therapies known in the art and appropriate for the neurological dysfunction to be treated or prevented.
[00228] The anti-BBB-R antibody of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and if desired, for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. The dosage can be by any suitable route, for example, by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is short or continuous. Various dosage schedules including, but not limited to, single or multiple administrations at various points in time, bolus administration and pulse infusion are contemplated in the present application.
[00229] Antibodies of the invention are formulated, dosed and administered in a manner consistent with good medical practice. Factors for consideration in this context include the specific dysfunction to be treated, the specific mammal to be treated, the clinical condition of each patient, the cause of the dysfunction, the location of the agent's distribution, the method of administration, the administration schedule and other factors known to medical specialists. The antibody does not need to be, but is optionally formulated with one or more agents currently used to prevent or treat the dysfunction in question or to prevent, mitigate or ameliorate one or more side effects of administering the antibody. The effective amount of these
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136/171 other agents depends on the amount of antibody present in the formulation, the type of dysfunction or treatment and the other factors discussed above. These are generally used in the same dosages and with administration routes as described in the present application, or about 1 to 99% of the dosages described in the present application, or in any dosage and by any route that is empirically / clinically determined to be appropriate.
[00230] For disease prevention or treatment, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and disease course, whether the antibody is administered for preventive or therapeutic purposes, prior therapies, the patient's medical history and response to the antibody, and the physician's diagnosis. The antibody is properly administered to the patient at once or over a series of treatments. Depending on the type and severity of the disease, about 1 pg / kg to 15 mg / kg (eg 0.1 mg / kg to 10 mg / kg) of antibody may be an initial candidate dosage to administer to the patient, for example example, either by one or more separate administrations or by continuous infusion. A typical daily dosage can range from about 1 pg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, treatment would generally be continued until a desired suppression of the symptoms of the disease occurs. An exemplary dosage of the antibody would be in the range of about 0.05 mg / kg to about 10 mg / kg. In this way, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) can be administered to the patient. These doses can be administered intermittently, for example, every week or every three
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137/171 weeks (for example, so that the patient receives about two to about twenty or, for example, about six doses of the antibody). A higher initial loading dose can be administered, followed by one or more lower doses. However, other dosage regimens can be useful. It will be appreciated that a method to reduce the impact on the reticulocyte population by administering anti-TfR antibodies is to modify the amount or timing of doses, so that, in general, smaller amounts of circulating antibodies are present in the bloodstream to interact with reticulocytes . In a non-limiting example, a lower dose of anti-TfR antibodies can be administered more frequently than a larger dose would be. The dosage used can be balanced between the amount of antibody required to be delivered to the CNS (itself related to the affinity of the antigen-specific portion of the antibody in the CNS), the affinity of the antibody to TfR, and whether compounds protect them or not. red blood cells (ie, reticulocytes), stimulate growth and development, or inhibit the complement pathway are being co-administered or in series with the antibody. The progress of this therapy is easily monitored by conventional techniques and assays, as described in the present application and as known in the art.
[00231] It is understood that any of the above therapeutic formulations or methods can be performed with the use of an immunoconjugate of the invention in place of, or in addition to, an antibiotic antibody
D. Fabrication Items [00232] In another aspect of the invention, an article of manufacture is provided which contains materials useful for the treatment, prevention and / or diagnosis of the disorders described above. The article of manufacture comprises a container and a label, or package insert associated with the container.
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Suitable containers include, for example, bottles, vials, syringes, bags for IV solution, etc. Containers can be manufactured from a variety of materials such as glass or plastic. The container contains a composition that is by itself or combined with another composition, effective for treating, preventing and / or diagnosing the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a bottle that has a pierceable cap for a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used to treat the recommended condition. In addition, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises an additional cytotoxic agent or otherwise a therapeutic agent. The article of manufacture in that embodiment of the invention may, furthermore, comprise a package insert indicating that the compositions can be used to treat a specific condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), buffered saline phosphate, Ringer's solution and dextrose solution. In addition, it may include other materials desirable from a commercial and user point of view, including other buffers, thinners, filters, needles and syringes.
[00233] It should be understood that any of the articles of manufacture above may include an immunoconjugate of the invention in place of, or in addition to, an anti-BBB-R antibody.
[00234] The article of manufacture optionally also includes a package insert with instructions for treating a neurological dysfunction in a
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139/171 subject, where instructions indicate that treatment with the antibody, as disclosed in the present application, treats neurological dysfunction, and optionally indicates that the antibody has enhanced absorption through BBB due to its low affinity for BBB-R .
Examples
Example 1
Generation and Characterization of Low Affinity Anti-TfR Arts Bodies [00235] The field recognized that the transferrin receptor's (TfR) natural ability to transport transferrin across the blood-brain barrier (BBB) can be exploited to allow the transport of heterologous molecules in the brain from the bloodstream (see, for example, WO 9502421). Applicants previously developed an important modification of this system, (Sei. Transi. Med. 3, 84ra43 (2011)) in other words, that the transport to the brain and the retention in the brain of a heterologous molecule conjugated to an anti- transferrin (anti-TfR) was substantially improved by decreasing the affinity of anti-TfR with the transferrin receptor, within a certain range.
[00236] A panel of anti-TfR antibodies was generated with a progressive decrease in affinities for murine TfR, three of which (designated anti-TfR ^A , anti-TfR ^D , and anti-TfR ^E ) were later modified into one bispecific shape with another antibody arm being specific for BACE1. Each monospecific and bispecific antibody was evaluated in a competition ELISA assay for its affinities for murine TfR. Briefly, the assays were performed on maxisorp plates (Neptune, NJ) with a 2.5 pg / ml layer of hexahistidine-labeled purified muTfR (muTfR-His) in PBS at 4 ° C ^and overnight. The plates were washed with 0.05% PBS / Tween 20 and blocked using buffer
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140/171 super-blocking block in PBS (Thermo Scientific, Hudson, NH). A serial bivalent 1: 3 IgG (anti-TfR ^A , anti-TfR ^D , anti-TfR ^E ) or bispecific Ab (anti-TfR ^A / BACE1, anti-TfR ^D / BACE1, or anti-TfR ^E / BACE1 ) fol combined with 1nM biotinylated anti-TfR ^A and added to the plate for 1 hour at room temperature. The plates were washed with 0.05% PBS / Tween 20 and HRP-streptavidin (SouthernBiotech, Birmingham) was added to the plate and incubated for 1 hour at room temperature. The plates were washed with 0.05% PBS / Tween 20. anti-TfR ^A biotinylate bound to the plate was detected using ato substrate (BioFX Laboratories, Owings Mills). (Figure 1 A). The IC50 values observed for the binding of each monospecific or bispecific antibody to the murine TfR in the assay are shown in Table 2.
Table 2
IC50 Values for Antibody Binding by ELISA Competition
Antibody IC50 TfR ^A 1 nM TfR ^D 66 nM Tf _R E 20 μΜ TíR ^a / BACE1 14 nM Tyr ^D / BACE1 1.6 μΜ TíR ^e / BACE1 95 μΜ
[00237] Distribution of the antibody after a single administration in mice was performed as follows. Female wild type C57B / 6 mice aged 6 to 8 weeks were used in all studies. Animal care was in accordance with institutional guidelines. The mice were injected intravenously with 50 mg / kg or a control IgG, anti-BACE1 or an anti-TIR / BACE1 variant. The total injection volume did not exceed 250 µL and the antibodies were diluted in D-PBS when necessary (Invitrogen). After the indicated time, the mice were perfused with D-PBS at a rate of 2
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141/171 mL / min for 8 minutes. The brains were extracted and the cortex and hippocampus isolated, homogenized in 1% NP-40 (Cal-Biochem) in PBS containing EDTA Mini Complete Complete protease inhibitor cocktail tablets (Roche Diagnosis). The homogenized brain samples were rotated at 4 ^S C for 1 hour before turning at 14,000 rpm for 20 minutes. The supernatant was isolated for the measurement of the brain antibody. Whole blood was collected prior to perfusion in microtainer tubes with EDTA (BD Diagnostics), allowed to sit for 30 minutes at room temperature and turned down at 5000x g for 10 minutes. An upper layer of plasma was transferred to new tubes for measurement of antibodies and mice.
[00238] Total antibody concentrations in the plasma of rats and brain samples were measured using an antihuFc / anti-huFc ELISA. Maxisorp NUNC 284 well immunoplates (Neptune, NJ) were coated with F (ab ') 2 fragments of donkey anti-human IgG and Fc fragment specific polyclonal antibody (Jackson ImmunoResearch, West Grove, PA) overnight at 4 ^S C. The plates were blocked with PBS, 0.5% BSA for 1 hour at 25 ^S C; Each antibody (control IgG, anti-BACE1, and bispecific anti-TfR / BACE1 variants) were used as a standard to quantify the respective antibody concentrations. The plates were washed with PBS, 0.05% Tween-20 using a microplate washer (Bio-Tek Instruments, Inc., Winooski, VT), and standards and samples diluted in PBS containing 0.5% BSA, 0.35 M NaCl, 0.25% CHAPS, 5 mM EDTA, 0.05% Tween 20 and 15 ppm ProClin®is (Sigma-Aldrich) were added for two hours at 25 C. binding of antibody was detected with IgG goat anti-human F (ab ') s conjugated to horseradish peroxidase and an Fc specific polyclonal antibody (Jackson ImmunoResearch). The samples were developed using 3,3 ', 5,5'-tetramethylbenzidine (TMB) (KPL, Inc., Gaithersburg, MD) and the absorbance measured on a 450 nm Multiskan reader
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Ascent (Thermo Scientific, Hudson, NH). Concentrations were determined from the standard curve using a four-parameter nonlinear regression program. The assay showed lower quantification limit values (LLOQ) of 3.12 ng / mL in serum and 12.81 ng / g in the brain. The statistical analysis of the differences between the experimental groups was performed using a two-tailed unpaired t test.
[00239] The results are shown in Figures 1B and 1 D. Both the control IgG and the anti-BACE1 antibody showed limited absorption in the brain, which persisted over the 10-day measurement period, while their plasma concentrations were higher than any of the molecules tested at all points in time, despite gradual clearance over time. Of the three anti-TfR / BACE1 variants evaluated, both anti-TfR ^A / BACE1 and anti-TfR ^D / BACE1 demonstrated concentrations between 35 and 40 nM in the brain and 1 day after the dose (7 to 8 times higher than IgG control, Figure 1D). However, the concentration of anti-TfR ^A / BACE1 in the brain decreased rapidly after day 2 and returned to control levels on day 6. Anti-TfR ^D / BACE1 persisted longer in the brain than anti “TfR ^A / BACE1 , with a more gradual decline in brain concentrations; however, by day 10 the concentration combined with the control. Anti-TfR ^E / BACE1 showed a much more moderate entry into the brain (2 to 3 times the control), but declined over the following days, which was much less than that of the other two variants of the antibody. Plasma levels of all three antibody variants (Figure 1B) have declined over time. AntiTfR ^A / BACE1 was completely released from plasma by day 4, while anti-TfR ^D / BACE1 was fully released by day 10 and anti-TfR ^E / BACE1 still persisted in plasma at a level comparable to the control IgG or to antiBACE1.
[00240] Taken together, these findings were
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143/171 consistent with the previous finding that a reduction in the affinity of an antibody to TFR actually improves its retention in the brain, as the higher affinity antibody used (anti-TfR ^A / BACE1) was released more quickly than brain and the least affinity antibody used (anti-TfR ^E / BACE1) persisted longer in the brain. However, it was also clear from the data that the total amount of anti-TfR ^D / BACE1 that was transported to the brain over time was much greater than that of antiTfR ^E / BACE1, suggesting that there is an optimal affinity between the anti- TfR ^D / BACE1 and the anti-TfR ^E / BACE1 to maximize both transport through BBB and persistence in the brain.
[00241] The presence and persistence of the transported molecule in the brain and plasma is only a measure of potential effectiveness; of further interest is the activity of the molecule in these compartments. Consequently, the enzymatic activity of BACE1 was tested in both compartments, by measuring the amount of Αβ-Μο (a by-product of dividing the enzymatic activity of BACE1 on the amyloid precursor protein (APP)). Briefly, antibody treatment and infusions were performed on wild type mice as specified above. For Αβ- | measurements. _4Ο cerebral hemispheres were homogenized in guanidine hydrochloride buffer and the samples rotated for 3 hours at room temperature before dilution (1:10) in 0.25% casein, 5mM EDTA (pH 8.0) in PBS containing aprotinin ( 20mg / mL) and leupeptin (10mg / mL) just added. Diluted homogenates were rotated down at 14,000 rpm for 20 min. and the supernatants were isolated for measurement of Αβι_ ₄₀ . The plasma was prepared as described above. The concentrations of Αβ ₁ . ₄₀ total rat in plasma and brain were determined using a sandwich ELISA following procedures similar to those described above. Cerebral hemispheres for measuring _{Αβ ν40}
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144/171 were homogenized in 1% NP-40 (Cal-Biochem) and rotated for 1 hour at room temperature before turning at 14,000 rpm for 20 minutes. Rabbit polyclonal antibody specific for the C-termination of Αβι- ₄ ο (Millipore, Bedford, MA) was coated on the plates and the biotinylated anti-mouse monoclonal antibody M3.2 Αβ (Covance, Dedham, MA) was used for detection. The assay showed LLOQ values of 1.96 pg / mL in the plasma and 39.1 pg / g in the brain. The statistical analysis of the differences between the experimental groups was performed using a two-tailed unpaired t test.
[00242] The results for plasma and brain are shown in Figures 1C and 1E, respectively, being consistent with the amount of antibody present in each compartment at the indicated time (see Figures 1B and 1D). Importantly, the amount of Αβ-Μο observed in the brain over time was the lowest over the longest period in mice treated with anti-TfR ^D / BACE1.
Example 2a
Effect of Αντι-TfR Dosage on Reticulocytes [00243] Unexpectedly, in the treatment of mice with monospecific anti-TfR ^A or anti-TfR ^D , all dose levels of 1 mg / kg or greater, clinical and acute signs were observed that were not seen in mice treated with bispecific anti-TfR ^A / BACE1 or antiTfR ^D / BACE1 (see Table 3).
Table 3
Symptoms Observed in Mice After Antibody Administration
Antibody Dose (mg / kg) Acute Clinical Signs Control IgG (isotype 50 * none
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Antibody Dose(mg / kg) Acute Clinical Signs Combined) Anti-TfR ^D (comprising effector function) 0.01 * none 0.1 1 Deep lethargy after dose within 5 minutesOccasional spasmodic movements in some animals.Disorganized appearance, arched for 20 to 25 minutes post-dose.Red urine seen in some miceCompletely reversible within hours 5 25 50 Anti-TfR ^c 7Bace (not including effector function) 1* none 5 * 25 50 200
[00244] ^A Reticulocyte decrease was not observed at these dose levels [00245] Specifically, monospecificly treated mice exhibited lethargy after the dose within 5 minutes of treatment, where they became immobile and unresponsive (with occasional spasmodic movements in some animals), followed by the development of a disorganized appearance, arched for 20 to 25 minutes after the dose. All of these observed effects disappeared within hours after treatment. Certain antibody-treated mice
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146/171 monospecific also appeared to present the occasional presence of blood in the urine, as well as apparent hypotension within 1 hour after the dose, based on the difficulty in collecting terminal cardiac blood compared to the collection in animals treated with the bispecific. Because it is known that the immature red blood cells of mice express TfR (see Figure 2A), existing in the peripheral bloodstream and the effects seen in mice can be explained if such blood cells have been injured, the impact of antibody treatment on red blood cells immature (reticulocytes) was investigated in mice.
[00246] The mice were dosed intravenously with a single injection of 1 mg / kg, 5 mg / kg, or 50 mg / kg of anti-TfR ^D or antiTfR ^D / BACE1, or with a single injection of IgG control of 50 mg / kg, using the same procedure as described in Example 1, and whole blood samples were taken 1 hour post-dose and placed in collection tubes containing potassium EDTA. Red blood cell and reticulocyte counts and indices were determined in these blood samples using Sysmex XT2000ÍV (Sysmex, Kobe, Japan), according to the manufacturer's instructions. Briefly, Sysmex detects and classifies the total reticulocytes, as well as the fraction of immature reticulocytes (sum of high and medium / intermediate fluorescent reticulocytes), by flow cytometry, using a fluorescent polymethylene dye by binding to cellular RNA and measuring the resulting cell light scattering characteristics.
[00247] At 1 hour post-dose, anti-TfR ^D reduced the levels of immature reticulocytes at all dose levels tested, approximately to the same extent, regardless of dose. Mice treated in each anti-TfR ^D dosing group also demonstrated acute clinical signs of similar severity and penetrance (see Figure 2B). In contrast, blood samples from mice treated with 1 mg / kg and 5
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147/171 mg / kg of anti “TfR ^D / BACE1 showed similar fractions of immature reticulocytes as those samples treated with control IgG. Mice treated with 50 mg / kg of anti-TfR ^D / BACE1 demonstrated a marked reduction in reticulocytes (for about 50% of control quantities) (Figure 2B), but this reduction was not accompanied by any acute clinical signs. Thus, the bispecific antibody containing antiTfR ^D has less impact on reticulocyte levels than monospecific anti-TfR ^D and does not obtain acute adverse clinical signs.
[00248] The experiment was repeated, then including a second bispecific antibody of a different affinity for TfR. The mice were dosed intravenously with a single injection of 5 mg / kg, 25 mg / kg, or 50 mg / kg of anti-TfR ^A or anti-TfR ^D / BACE1, or with a single injection of 50 mg / IgG control kg, using the same procedure as described in Example 1, and whole blood samples were taken 24 hours and 7 days after the dose. Reticulocyte counts were measured in whole blood as described above. The results are shown in Figure 2C. At 24 hours post dose, all samples of rats treated with anti “TfR ^A / BACE1 demonstrated similar marked reductions in the total reticulocyte count. Samples treated with 25 mg / kg and 50 mg / kg of antiTfR ^D / BACE1 demonstrated similarly low reticulocyte counts as samples treated with anti-TfR ^A / BACE1. However, samples treated with 5 mg / kg of anti-TfR ^D / BACE1 demonstrated only a modest reduction in the number of relative reticulocytes with the IgG control sample at 24 hours post dose. After 7 days of the dose, all groups showed normal levels of reticulocytes (Figure 2C), suggesting recovery from the initial depletion of reticulocytes, with the exception of the 50 mg / kg sample of antiTfR ^D / BACE1, which demonstrated a sustained reduction in levels of reticulocytes (approximately 50%) relative to control quantities.
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Thus, only the lowest tested dose of anti-TfR ^D / BACE1 had a moderate impact on reticulocytes, while all other doses tested led to an almost complete loss of reticulocytes within 24 hours after the dose, indicating that the reduction antibody affinity (anti-TfR ^D relative to anti-TfR ^A ) and dose alleviate safety concerns related to reticulocyte loss. At 7 days post-dose, however, only the highest dose of anti-TfR ^D / BACE1 had any measurable impact on reticulocyte levels, while all other doses tested demonstrated a recovery of reticulocyte counts to levels similar to those of the IgG control mice. Notably, the absolute affinity of the antibody to TfR at 7 days after the dose was not as important as the persistence of the antibody in the bloodstream for the most important time points. Regardless of the much greater affinity of anti-TfR ^A 7BACE1 with TfR (Table A), mice treated with high doses of anti-TfR ^A 7BACE1 demonstrated a recovery in reticulocyte numbers within 7 days, which corresponded with the faster release of this antibody a from the circulation relative to anti-TfR ^D / BACE1 (as shown in Example 1, Figure 1B).
[00249] To the extent that a dose response was observed in reticulocyte depletion, experiments were performed to determine whether it was possible to correlate various dose levels with an associated ability to reduce Abeta in the brain. Briefly, female C57B / 6 wild type mice aged 6-8 weeks were used in all studies. Mice were injected intravenously with 50 mg / kg or control IgG, or anti-TfR / BACE1. The total injection volume did not exceed 250 pL and the antibodies were diluted in D-PBS (Invitrogen) when necessary. After the indicated time, the mice
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149/171 were perfused with D-PBS at a rate of 2mL / min for 8 minutes. The brains were extracted and the cortex and hippocampus isolated, homogenized in 1% NP-40 (Cal-Biochem) in PBS containing EDTA Mini Complete Complete protease inhibitor cocktail tablets (Roche Diagnosis). The homogenized brain samples were rotated at 4 ^to C for 1 hour before turning at 14,000 rpm for 20 minutes. The supernatant was isolated for the measurement of the brain antibody. Whole blood was collected prior to perfusion in microtainer tubes with EDTA (BD Diagnostics), allowed to sit for 30 minutes at room temperature and turned down at 5000x g for 10 minutes. An upper layer of plasma was transferred to new tubes for measurement of antibodies and Αβ-Μοόθ mice.
[00250] Total antibody concentrations in the plasma of rats and brain samples were measured using an antihuFc / anti-huFc ELISA. Maxisorp NUNC 384 well immunoplates (Neptune, NJ) were coated with F (ab ') ₂ fragments of donkey anti-human IgG and Fc fragment specific polyclonal antibody (Jackson ImmunoResearch, West Grove, PA) overnight at 4 ^S C. the plates were blocked with PBS, 0.5% BSA for 1 hour at 25 C; Each antibody was used with a standard to quantify the respective antibody concentrations. The plates were washed with PBS, 0.05% Tween-20 using a microplate washer (Bio-Tek Instruments, Inc., Winooski, VT), and standards and samples diluted in PBS containing 0.05% BSA, 0.35 M NaCl, 0.25% CHAPS, 5 mM EDTA, 0.05% Tween-20 and 15 ppm ProClin®is were added for two hours at 25 C. binding of antibody was detected with anti-IgG goat human conjugated with horseradish horseradish peroxidase, Fc-specific polyclonal antibody, developed using 3,3 ', 5,5'-tetramethylbenzidine (TMB) (KPL, Inc., Gaithersburg, MD) and the absorbance measured in a 450 nm Multiskan Ascent reader (Thermo Scientific, Hudson, NH). Concentrations
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150/171 were determined from the standard curve using a four-parameter nonlinear regression program. The assay showed lower quantification limit values (LLOQ) of 3.12 ng / mL in serum and 12.81 ng / g in the brain. The statistical analysis of the differences between the experimental groups was performed using a two-tailed unpaired t test.
[00251] Abetai-40 has also been detected in the brain and plasma. Briefly, mice were treated with antibody and perfused according to the method described above. For measurements of hemβι- ₄₀ brain hemispheres, they were homogenized in 5M guanidine hydrochloride buffer and the samples rotated for 3 hours at room temperature before dilution (1:10) in 0.25% casein, 5mM EDTA (pH 8 , 0) in PBS containing freshly added aprotinin (20 mg / ml) and leupeptin (10 mg / ml). Diluted homogenates were rotated downward at 14,000 rpm for 20 min. and the supernatants were isolated for Αβι measurement. _4th . The plasma was prepared as described above. The concentrations of total rat Αβν ₄₀ in plasma and brain have been described using a sandwich ELISA following procedures similar to those described above. Rabbit polyclonal antibody specific for the C-termination of Αβ- | .. ₄ ο (Millipore, Bedford, MA) was coated on the plates and the biotinylated anti-mouse monoclonal antibody M3.2 Αβ (Covance, Dedham, MA) was used to detection. The assay showed LLOQ values of 1.96 pg / mL in the plasma and 39.1 pg / g in the brain. The statistical analysis of differences between experimental groups was performed using a two-tailed unpaired t test.
[00252] A robust and sustained reduction in cerebral Abeta, both at dose levels of 25 and 50 mg / kg for anti-TfR ^D / BACE1 was observed (Figure 2D), while anti-TfR ^A / BACE1 demonstrated a robust, but acute reduction in cerebral Abeta at all three dose levels (Figure 2E). These data were considered with the pharmacokinetics
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151/171 observed of the compounds both in the periphery and in the brain (Figure 2F-2H). From these data, it was apparent that a dosage level of 25 mg / kg of anti-TfR ^D / BACE1 is sufficient to significantly reduce the levels of cerebral Abeta in these studies.
[00253] Reticulocyte depletion by anti-TfR antibody species may be due to a variety of different natural processes, including effector / antibody dependent cell-mediated cytotoxin (ADCC), complement-dependent cytotoxin (CDC), lysis / apoptosis mediated by direct target, and / or phagocytosis of reticulocytes opsonized by macrophages. A series of experiments was carried out to better understand the mechanisms responsible for the reticulocyte depletion observed after administration of anti-TfR antibody.
Example 2b
Impact of Modulating Effective Function [00254] In addition to differing in affinity and valence for TfR, the monospecific and bispecific anti-TfR antibodies used in previous experiments also differed in the degree of their effector functions. Monospecific anti-TfR antibodies were produced in CHO cells and had mammalian glycosylation and wild-type effector function. The anti-TfR / BACE1 antibodies showed a severely reduced or eliminated ability to interact with Fcy receptors with the use of one or more of the following methods of known techniques: Cancellation of glycosylation due to the presence of an N297G or N297A mutation in the Fc region (Atwal etal., Sei. Transi Med. 3, 84ra43 (2011); Fares Al-Ejeh et al., Clin. Cancer Res. (2007) 13: 5519s-5527s), modification of the antibody Fc region to contain an aspartic acid for the alanine mutation at position 265 (D265A), known to completely nullify the effector function (see, for example, US patent 7,332,581), or by producing the antibody in a way that prevented
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152/171 wild-type mammalian glycosylation, as in E. coli production.
[00255] The studies in rats performed in Example 2A were repeated with these antibodies modified for Fc and also in different strains of rats, or lacking the Fcy receptors or the C3 complement, to evaluate the potential mechanisms of depletion of reticulocytes, including ADCC or effector-oriented CDC, respectively; whole blood samples were assessed by total reticulocyte counts 24 hours after intravenous injection of the antibody. In the first experiment, administration of a 1 mg / kg or 25 mg / kg of monospecific anti-TfR ^D lacking the effector function for wild type mice had the same depletive effect on reticulocyte counts as an anti-TfR ⁰ antibody with effector function. total (compare Figure 3A with Figure 2B). However, no acute clinical signs were observed in mice treated with anti-TfR ^D antibody without effector, in sharp contrast to those treated with effector positive anti-TfR ⁰ antibody (Example 2A). Similarly, when effector-positive anti-TfR ^D was administered to mice lacking the Fcy receptor (to eliminate ADCC mechanisms that can be triggered by the effector function), reticulocyte levels were reduced to almost zero after a 25 mg / dose. kg, but no acute clinical signs were observed (Figure 3B).
[00256] The impact of bispecific anti-TfR ^D / BACE1 D265A antibodies lacking the effector function on reticulocyte levels was also assessed in Fcy knockout mice (Figure 3B). Even the complete annulment of the effector function of the antibody and the absence of Fcy receptor in the mice did not mitigate the depletion of reticulocytes when administered at a dose level of 25 mg / kg. Consistent with other experiments with the use of anti-TfR / BACE1 antibodies without effector, bispecific in wild type mice, no adverse clinical signs were observed in
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153/171 treated Fcy knockout mice.
[00257] To determine if the presence of the effector function is sufficient to lead to acute clinical symptoms, and later characterize the contribution of the effector function to the depletion of reticulocytes, the experiments were repeated in wild type mice comparing a low dose (5 mg / kg) of anti-TfR ^D / BACE1 D265A without effector with an equivalent dose of anti-TfR ^D / BACE1 positive for total effector (Figure 3C). Acute clinical signs were observed in the introduction of the effector function in the bispecific antibody. In addition, robust reticulocyte depletion was observed with positive antibodies to the effector at the lowest dose level relative to the non-effector version of the antibody (Figures 3C and 2C). From these combined data, the effector function is not necessary to direct depletion of reticulocytes, but it visibly contributes to this depletion, particularly at lower dose levels. Importantly, the acute clinical symptoms seen in mice are linked to the effector status of the antibody, so that antibodies without effector or Fcy knockout mice can both completely mitigate these symptoms.
[00258] To determine whether the complement cascade was involved in either the clinical symptoms or the loss of reticulocytes, the experiments were performed again on mice deficient in the C3 complement (for example, mice missing the normal complement cascade). As shown in Figure 3D, effector-positive anti-TfR ^A caused both deep reticulocyte depletion and robust acute clinical symptoms in these mice, indicating that the C3 complement and the associated complement cascade do not play a major role in the conduction or observed effects. when the administered antibody has full effector function. To test whether the same results could be obtained in the absence of full effector function, knockout C3 mice were dosed
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154/171 with anti-TfRD / BACE1 antibodies without effector to determine whether complement measured residual reticulocyte depletion. The results are shown in Figure 3E. In fact, residual reticulocyte depletion is recovered when both the effector function and the complement cascade are eliminated by dosing in knockout C3 mice with bispecific anti-TfR antibodies without effector at high therapeutic dose levels (50 mg / kg). Thus, the complement appears to act as a depletion mechanism for reticulocytes followed by the administration of anti-TfR antibodies without effector in mice.
[00259] An in vitro complement-dependent cytotoxin (CDC) assay was also performed. In summary, CDC assays were performed using primarily rat bone marrow cells or rat erythroleukemic lymphoblasts (HPA Cultures, UK) as target cells and rabbit serum-derived complement (EMD Chemicals, Gibbstown NJ). The cells were counted and viability determined on Vi-Cell ™ (Beckman Coulter, Fullerton, CA). Anti-TfR ^A / BACE1, anti-TfR ^A antibodies or negative or positive control antibodies (IgG or anti-H2Kb, respectively) were serially diluted in assay medium (RPMI-1640 medium supplemented with 20 mM HEPES, pH 7, 2 and 1% FBS) and distributed on a white, 96-well flat-bottom tissue culture plate (Costar; Corning, Acton MA). After addition of serum complement diluted 1: 3 in the assay medium and target cells (2 x 10 ⁵ cells / well), the plate was incubated with 5% CO ₂ for 2 hours at 37 C. Plates then they were left at room temperature for 10 minutes with constant stirring. The extent of cell lysis was quantified by measuring the luminescence intensity with a SpectroMax ™ M5 plate reader. The luminescence values of the sample dilutions were plotted against the antibody concentration and the dose response curves were adapted to a four-parameter model using the
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Graph Pad ™ (Graph Pad Software Inc.).
[00260] Interestingly, neither the competent monospecific anti-TfR ^A of effector function nor the bispecific anti-TfR ^A / BACE1 without treatment effector of mice cells in the presence of serum complement resulted in complement-mediated lysis of the cells, while the positive anti-H2Kb control demonstrated significant cell lysis (Figure 4A). In particular, the differentiating activity of the antibody effector did not appear to influence its ability to remove CDC activity. A non-limiting explanation is that the complement can mediate reticulocyte depletion in vivo through opsonization of circulating reticulocytes through splenic and hepatic macrophages (Garratty (2008), Transfusion Med. 18 (6): 321-334; Mantovani et al, (1972) J. Exp. Med. 135: 780-792; Molina et al., (2002) Blood 100 (13): 4544-4549), a mechanism that must remain intact with fragments of anti-TfR F (ab ' ) ₂ .
[00261] Similar in vitro experiments have also been carried out to confirm the in vivo results described earlier, supporting a link between antibody-dependent cell mediated cytotoxin effector function (ADCC), acute clinical symptoms and reticulocyte depletion. ADCC assays were performed using PBMCs freshly isolated from healthy donors such as effector cells and cells from the primary bone marrow of rats or rat erythroleukemic lymphoblasts (HPA Cultures, UK) as target cells. To minimize donor variations from allotypic differences at position 158 of FcyRIIIA residue, blood donors were limited to those carrying the heterozygous FcyRIIIA genotype (F / V158). Briefly, PBMCs were isolated by density gradient centrifugation using a Uni-Sep blood separation tube (Accurate Chemical &Scientific; Westbury, NY). The target cells were pre-labeled with 1.4 mM solution of AM calcein
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156/171 (Molecular Probes) and were seeded in a 96-well round bottom plate (BD Biosciences; Mississauga, Ontario; Canada) at 4 x 10 ⁴ / well. Serial dilutions of anti-TfR / BACE1, anti-TfR and control antibody were added to plates containing the target cells, followed by incubation at 37 ^S C with 5% carbon dioxide for 30 minutes to allow opsonization. Final antibody concentrations ranged from 1,000 to 0.004 ng / mL after 4-fold serial dilutions. After incubation, 1 x 10 ^b PBMC of effector cells in 100 µL of assay medium was added to provide a 25: 1 ratio of effector to target cells and the plates were incubated for an additional 3 hours. The plates were centrifuged at the end of the incubation and the fluorescent signals in the supernatants were measured using a SpectraMax ™ M5 microplate reader, with excitation at 485 nm and emission at 520 nm. The signals from wells containing only the target cells represented the spontaneous release of AM calcein from the labeled cells (spontaneous release), while the wells containing target cells dissolved with Triton ™ X-100 provided the maximum signal available (maximum lysis). Antibody-independent cellular cytotoxin (AICC) was measured in wells containing target and effector cells without the addition of antibodies. The specific ADCC extension was calculated as follows:
[00262]% ADCC - 100 x (Sample signal - AICC): · (maximum lysis - spontaneous release) [00263] The ADCC values of the sample dilutions were plotted against the antibody concentration and the dose response curve has been adapted to a four parameter model using Graph Pad ™ (Graph Pad Software Inc.).
[00264] The anti-TfR ^A used in this assay had an effective action, while the anti-TfR ^A 7BACE1 used in the assay had no effector function. As shown in Figure 4B, the antibody with effector function induced
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ADCC, while the anti-TfR ^A / BACE1 antibody with effector function is not missing, correlating with the results of previous experiments in rats. These data further support the idea that acute clinical signs in treated mice are due to the ADCC activity produced by the positive effector antibodies binding to circulating reticulocytes, and the effector-driven ADCC may also contribute to the reticulocyte depletion following administration of antibodies (Figure 3C).
Example 2c
Impact of Modulation or BACE1 Binding Fc [00265] The role of the Fc arm and the BACE1 arm were examined separately for their potential involvement in mediating reticulocyte depletion. Monospecific and bispecific anti-TfR with wild-type IgG1 Gc regions having full effector function and normal glycosylation were generated. Briefly, TfR (orifice) and IgG (protuberance) semi-antibodies were expressed separately in CHO and annealed in vitro, as described (Carter, P. (2001) J. Immunol. Methods 248, 7-15: Ridgway, JB, Presta , LG, and Carter, P. (1996) Protein Eng. 9, 617-621; Merchant, AM, Zhu, Z., Yuan, JQ, Goddard, A., Adams, CW, Presta, LG, and Carter, P (1998) Nat. Biotechnol. 16, 677-681; Atwell, S., Ridgway, J. B „Wells, JA, and Carter, P. (1997) J. Mol. Biol. 270, 26-35). F (ab ') ₂ fragments were generated from anti-TfR IgG, antiTfR / IgG or anti-TfR / BACE1 antibodies by digestion with immobilized pepsin. The antibody was reconstituted in 100 mM sodium acetate, pH 4.2 and incubated with immobilized pepsin resin (0.3 ml settled gel / mg IgG) overnight at 37 ^Q C with rotation. After incubation, the sample was centrifuged to separate the immobilized pepsin from the digested F (ab ') ₂ mixture. The F (ab ') s fragment was then purified using SP sepharose, a strong cation exchange resin (1 mL HiTrap ™ column (Supelco)). The sample was loaded at 50 mM
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158/171 NaOAc pH 5.0 and eluted with a 0-0.5 M NaCI gradient over 20 column volumes, after which the sample was dialyzed against PBS, pH 7.4. Experiments on rats were performed with these antibodies and F (ab ') ₂ using the same procedures as above and an intravenous dose of 25 mg / kg of monospecific F (ab') ₂ or an intravenous dose of F (ab ') 2 bispecific or control, or antibody; whole blood samples were evaluated for total reticulocyte counts 24 hours after intravenous injection of antibody / F (ab ') 2. The results are shown in Figure 5A-5C.
[00266] Administration of anti-TfR ^D F (ab ') 2 has a reticulocyte depletion effect very similar to administration of anti-TfR ^D antibody (compare Figure 5A with Figures 3A and 3B), indicating that the Fc portion of the antibody it is not necessary for reticulocyte depletion observed at the dose levels assessed. Although bispecific F (ab ') 2 molecules demonstrate a slight attenuation of reticulocyte depletion relative to bispecific full-length IgG antibodies (compare Figure 5B with Figure 2C), it should be noted that this is more likely due to the faster release in general of F (ab ') ₂ relative to IgG (Covell et. AL, (1986) Cancer Res. 46: 3969-3978), leading to a reduced overall antibody exposure over the 24 hour post-dose interval. Nevertheless, reticulocyte depletion observed after administration of bispecific F (ab ') ₂ antibodies still emphasizes the conclusion that the Fc region is not necessary for reticulocyte depletion to occur. Bispecific antibodies missing from the BACE1 arm (antiTfR ^D / lgG control) depleted the reticulocytes to the same degree as the antiTfR ^D / BACE1 (Figure 5C), demonstrating that the BACE1 arm also does not contribute to the elimination of reticulocytes.
Example 3
Additional Link Affinity Elaboration [00267] Some of the results above suggested that there was
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159/171 an affinity and dose component of the observed degree of reticulocyte depletion (Figure 20). To better understand how affinity and dose impact reticulocyte depletion, the rat dosing experiments performed in Example 2 were repeated with additional low-activity anti-TfR antibodies, specifically anti-TfR ^E / BACE1 at two different dose levels (25 mg / kg and 50 mg / kg). Anti-TfR ^E at any of the doses tested essentially had no impact on reticulocytes (Figure 6A), while similar doses of anti-TfR ^A / BACE1 or antiTfR ^D / BACE1 depleted the reticulocytes. From the results discussed in Example 1, it was observed that anti-TfR ^E / BACE1 had better sustained plasma exposure and persistence in the brain, but less robust transport across the blood-brain barrier and then anti-TfR ^D / BACE1. Since administration of anti-TfR ^c 7BACE1 resulted in depletion of reticulocytes, but administration of anti-TfR ^E / BACE1 did not, anti-TfRs variants were generated with affinities among which anti-TfR ^D and anti-TfR ^E to TfR to see if the safety profile of the antibody can be improved without sacrificing BBB transport and persistence in the brain.
[00268] Briefly, site-directed mutagenesis was used to combine the two mutation points representing the anti-TfR ^D and anti-TfR ^B variants respectively into a single designated antiTfR ^Db antibody, using standard mutagenesis techniques. Similarly, the two mutation points representing the anti-TfR ^D and anti-TfR variants ^c respectively in a single designated anti-TfR ^Dc antibody. Both antibodies were produced in a bispecific format with anti-BACE1 using protrusion and orifice technology, as described in Example 2C. The affinities of both antibodies were between those of the anti-TfR ^D and anti-TfR ^ antibodies to TfR, and the anti-TfR ^Db / BACE1 antibody showed an approximately three times greater affinity for TfR than that of the antibody
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160/171 anti-TfR ^Dc / BACE1. The rat administration / reticulocyte depletion experiment was repeated with these new variants and the results are shown in Figure 6B. Both variants demonstrated markedly improved reticulocyte depletion than anti-TfR ^D / BACE1 antibody at the same dose level and reticulocyte levels approximately like those of mice treated for control within 24 hours of dosing. As expected, the plasma antibody concentration of both new antibody variants over time, the cerebral antibody concentration (both the maximum value and the decrease over time) and the Αβι reduction. _4Ο was between that of anti-TfR ^D / BACE1 and anti-TfR ^E / BACE1 when administered at the same dose levels.
[00269] The impact of affinity and dose on TfR expression in the blood-brain barrier were also examined. Mice were treated with a single dose of anti ~ TfR ^A / BACE1 or antiTfR ^D / BACE1 at 5, 25 or 50 mg / kg, and the expression of TfR in the brain was assessed at 4 days post dose via Western blot. The brains of the antibody-treated mice were perfused with PBS prior to extraction and the cortex and hippocampus isolated, homogenized in 1% NP-40 (Cal-Biochem) in PBS containing EDTA Mini Complete free protease inhibitor cocktail tablets ( Roche Diagnosis). The homogenized brains were related to 4 ^S C for 1 hour before turning at 14,000 rpm for 20 minutes. The supernatant was isolated and equal concentrations of protein were separated by 4-12% Novex Bis-Tris gel (Invitrogen). The membranes were incubated with anti-TfR antibodies (Invitrogen) and anti-actin (Abeam) overnight at 4 C, followed by secondary antibodies IRDye® (LiCor Biosciences) at room temperature for 2 hours. Immunoblots were photographed and bands were quantified by densitometry using the Odyssey Infrared Imaging System ™ (Li-Cor
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Biosciences, Lincoln, NE). Four days after the dose, TfR expression in all three samples treated with anti-TfR ^D / BACE1 was similar, although slightly depressed from the control levels at higher dose levels (Figure 6C). In contrast, increasing doses of antiTíR ^a / BACE1 antibody resulted in a considerable decrease in TfR expression in the blood-brain barrier 4 days after the dose. Thus, the reduction in the affinity of the anti-TfR antibody also improves the dose-dependent reduction in the expression of cerebral TfR, potentially further contributing to improving the overall safety profile of the antibody.
Example 4
BBB Permeability Assessment [00270] One concern of exploring a blood-brain barrier transport receptor for the transport of heterologous molecules to the brain is that the BBB itself may be impaired. Consequently, BBB permeability to antibodies at anti-TfR dosing was investigated. Wild-type mice were administered intravenously with 50 mg / kg of control IgG, or 25 mg / kg of each of the indicated combinations of co-injected antibodies. Average antibody absorption in the brain 24 hours after intravenous injection was assessed using a generic human Fc ELISA as per Example 1, or using an anti-BACE1 specific ELISA using procedures similar to those described in Example 1. The extracellular domain of BACE1 was used as the protein layer and detection was performed with goat anti-human IgG F (ab ') ₂ conjugated to horseradish peroxidase, Fc specific polyclonal antibody. This assay showed LLOQ values of approximately 2.56 ng / g for anti-BACE1 and 12.8 ng / g for antiTfR ^D / BACE1. Levels of cerebral Αβ- ^ ο were measured after administration using the same procedure shown in Example 1.
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162/171 [00271] The results are shown in Figures 7A - 7C. Exposure to brain antibody was the highest in control IgG + mice treated with anti-TfR ⁰ / BACE1, but also substantial in mice treated with combinations of antibodies containing anti-TfR ⁰ (Figure 7A). This correlates with the results in Example 1, in which the bispecific form of lower affinity of anti-TfR ⁰ is taken and persists in the brain for longer than the monospecific form of greater affinity of anti-TfR ⁰ Antibodies co-administered with anti-TfR antibodies were not taken in the brain in substantial amounts; the only anti-BACE1 observed in substantial amounts in the brain was that conjugated directly to anti-TfR ⁰ (Figure 7B). Similarly, the only activity of antiBACE1 observed was in mice treated with anti-TfR ^D / BACE1 (Figure 7C). Taken together, these data indicate that the blood brain barrier permeability to antibodies was not affected by the antiTfR treatment.
Example 5
Impact of Multiple Dosing on Reticulocyte Levels [00272] The previously mentioned studies focused on a single dose of anti-TfR antibody and the resulting impact on reticulocyte levels and concomitant acute clinical symptoms. In order to ascertain whether different effects were observed following multiple doses over a longer period of time, further studies were carried out. The same protocols, as described in the preceding examples, were used, except that, instead of a single intravenous dose, the mice were dosed intravenously once a week with 25 mg / kg of antiTfR ^D / BACE1 or an IgG control, for example. a total of four weeks; Tissue / blood was collected at 1.4 or 7 days after the second injection and after the fourth injection and processed using the protocols described above. Beyond
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163/171 of this, direct bilirubin, serum and total iron and binding capacity to unsaturated iron were determined for serum samples by colorimetric assays using Integra ™ 400 (Roche, Indianapolis, IN) according to the manufacturer's instructions. Six mice were used for each time point and treatment group.
[00273] The serum antibody concentration for antiTíR ^D / BACE1 was similar over time after 2 or 4 doses, suggesting that the release into the bloodstream of rats does not differ substantially after repeated dosing (Figure 8A). However, a slight decrease in overall antibody exposure was apparent 4 days after the fourth relative dose at the same time after the second dose, suggestive of the occurrence of rat anti-drug antibodies (ADAs) for the administered human IgG antibodies. Similar to serum antibody concentrations, brain antibody concentrations decreased for 4 days after the fourth dose, despite the persistence of antibodies present in the brain over time, mirroring what was observed after the second dose (Figure 8B). The plasma (Figure 8C) and brain (Figure 8D) levels of the well correlated with the observed amounts of antiTfR ^D / BACE1 present in the serum and brain after 2 or 4 doses.
[00274] Also relevant, no exacerbation of reticulocyte toxicity was observed in the multidose context. As shown in Figure 8E, the number of absolute reticulocytes improved dramatically from day 1 after the second dose to 7 days after the fourth dose (where the values returned to or exceeded the control levels). There was no evidence of a decrease in red blood cell mass or changes in serum iron or in total iron binding capacity (a surrogate parameter for serum transferrin) in four weeks. There was also no evidence of histopathological changes or altered levels of colorable iron in any evaluated tissues. Without being bound by theory, it is proposed that a
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164/171 improved bone marrow regenerative response obtained by administering the initial and sustained dose over the dosing period may be responsible for the improvement in the overall reticulocyte decrease observed after the fourth dose. In addition, the suspected presence of ADAs also reduced the general levels of circulating antibodies with repeated dosing, also contributing to the mitigation of reticulocyte depletion observed at week 4. Finally, the cerebral expression of TfR did not differ in mice treated with the anti- TfR ^D / BACE1 and the control IgG at 1.4 or 7 days after the fourth dose (Figure 8F).
Example 6
Impact of Erstroid Progenitor Cells Containing Effector and Biospecific Without Effector in Blood and Bone Marrow [00275] Additional experiments were carried out to elucidate the impact of the dosing antibodies on populations of erythroid progenitor cells in the bone marrow. First, to examine the time course of reticulocyte loss after anti-TfR / BACE1 measurement, blood and bone marrow were isolated at 1, 4, 16 and 24 hours after wild type mice were injected intravenously with 50 mg / kg of Control IgG or anti-TfR ^D / BACE1 lacking the effector function as a single bolus in 200 pL in sterile PBS (n = 6 / group). Blood and bone marrow were collected from animals at the indicated time points post-dose. Orbital bleeds were used for blood extraction after isoflurane and bone marrow anesthesia from a femur was harvested and single cell suspensions were prepared. The cells were then filtered through a 70 micron cell sieve. The cells were washed and resuspended in a defined volume of PBS. A fixed volume of cell suspension was added to a fixed concentration of fluorescent microspheres labeled with FITC and analyzed in a flow cytometer, collecting 5000
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165/171 microsphere events per sample to obtain cell counts. Quantitative analysis of erythroid populations was determined using flow cytometry. In both blood and bone marrow, distinct populations of erythroid cells were controlled through their expression of the Ter119 marker (a marker that was determined to be expressed only in mature murine erythrocytes and erythroid precursor cells), TfR expression and dispersion profile lateral (as previously described in Paniga et al., '' Expression of Prion Protein in Mouse Erythroid Progenitors and Differentiating Murine Erythroleukemia Cells. ”PLoS One 6, 9 (2011); Figs. 9A and 9B). Briefly, samples were incubated for 20 minutes on ice with anti-mouse Ter119-PE (eBioscience) and biotinylated anti-mouse TfR, followed by streptavidin eFluor450 (eBioscience). Samples were washed with PBS containing 0.5% BSA, 2mM EDTA and passed in a BD LSR Fortessa multicolored flow cytometer and analyzed using the FlowJo computer program (Ashland, OR).
[00276] Treatment with anti-TfR ^D / BACE1 lacking the effector function did not change the total number of erythrocytes in the blood compared to the control IgG (Fig. 9C), but it did however quickly and significantly reduce the circulation of reticulocytes expressing TfR in the blood (Fig. 9D). In contrast to the findings in the blood, anti-TfR ^D / BACE1 had no effect on any of the progenitor populations of erythroid cells in the bone marrow (Fig. 10CA-C), including populations with high TfR expression (EryA and EryB populations) ( Fig. 10B-C), and mature red cells negative for TfR (EryC population) (Fig. 10D). Together, these results demonstrated that anti-TfR ^D / BACE1 without effector only depletes reticulocytes expressing TfR in the blood in mice, without impacting other subpopulations of erythroid cells in the bone marrow after a single dose.
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166/171 [00277] To investigate the impact of total effector function, antibodies on erythrocyte subpopulations in both blood and bone marrow and to determine whether affinity plays a role in erythroid cell depletion, wild-type mice received a single IV dose 25 mg / kg anti-TfR ^A / BACE1 (Fc-), anti-TfR ^D / BACE1 (Fc-), anti-TfR ^D / BACE1 (Fc +), or control IgG (where “Fc-” indicates a antibody without effector due to the presence of D265A and N297G mutations or the lack of glycosylation and “Fc +” indicates an antibody with a wild-type effector function), following the same injection and sample collection process as above. Neither the presence of the effector function nor the affinity for TfR affected the total number of mature erythrocytes circulating in the blood after dosing antibodies, compared to the control IgG (Fig. 11A). Confirming the previous observation, dosing with anti-TfR / BACE1 antibodies without effector resulted in a rapid and prolonged decrease in reticulocytes expressing TfR in the blood (Fig. 11B, compare with Fig. 9D). In addition, TfR affinity did not change the extent to which bispecific antibodies led to loss of reticulocytes, as there were no significant differences over time or in the magnitude of reticulocyte decrease between animals dosed with anti-TfR ^Ã / BACE1 (Fc-) or anti-TfR ^D / BACE1 (Fc-) (Fig. 11B). However, dosing with anti-TfR ^D / BACE1 of full effector function (Fc +) resulted in an exacerbation of reticulocyte loss, as compared with bispecific antibodies without effector (Fig. 11B), suggesting that the effector function plays an important role on the severity of reticulocyte depletion after antibody measurement.
[00278] In the bone marrow, neither the bispecific antiTfR antibody without effector (Fc-) changed the total number of erythroid cells, compared to the control IgG (Fig. 11 A). However, anti-TfR ^D / BACE1 with full effector function (Fc +) reduced the total number of erythroid cells in 24 hours
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167/171 after the dose (Hg. 12A). Specifically, TfR positive erythroid precursor cells (EryA and EryB populations) were significantly and robustly reduced in the presence of full effector function, whereas anti-TfR / BACE1 antibodies without effector had no effect on subpopulations of erythroid cells positive for TfR compared to the control IgG. (Fig. 12B-C). Notably, the number of mature erythrocytes was transiently increased after dosing with anti-TfR ^D / BACE1 of full effector function (Fc +) in 4 to 16 hours after dose, compared with anti-TfR / BACE1 antibodies without effector ( Fc-) and the control IgG (Fig. 12D). In a non-limiting interpretation, this temporary increase may be due to a secondary compensation mechanism directing accelerated erythrocyte maturation in response to depletion of erythroid precursor cells. Taken together, these data suggest that an anti-TfR / BACE1 antibody without effector mitigates the loss of TfR-positive erythroid cells in the bone marrow.
Example 7
Impact of Monospecific and Bispecific Antibodies Containing Effector and Without Effector on a Human Erstroleukemin Cell Line and PRIMARY BONE MARROW MONONUCLEAR CELLS [00276] The examples mentioned above used anti-murine TfR antibodies, which do not specifically recognize human TfR. To make sure that the reticulocyte depletion observed in the studies in rats was exclusive to the murine system, further experiments were carried out using anti-TfR that binds to human TfR.
[00277] ADCC assays were performed using peripheral blood mononuclear cells (PBMCs) from healthy human donors as effector cells. A human erythroleukemin cell line (HEL, ATCC) and primary human bone marrow mononuclear cells (AllCells, Inc.) were used as target cells. To minimize the
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168/171 variability between donors that could potentially create allotopic differences at position 158 of residue in FcyRIIIA, blood donors were limited to those carrying heterozygous RcyRIIIA genotype (F / V158) in the first set of experiments (Figure 13A-B). For the second set of experiments (Figure 4A-B), only HEL cells were used as target cells, with PMBCs from healthy human donors carrying either the F / V 158 genotype or the FcyRIIIA V / V 158 genotype. The V / V158 genotype it was also included in this trial due to the known association with increased ADCC activity mediated by NK cells, as well as the ability to bind to IgG4 antibodies (Bowles and Weiner, 2005; Bruhns et al. 2008). The cells were counted and viability determined on Vi-Cell ™ (Beckman Coulter, Fullerton, CA), following the manufacturer's instructions.
[00278] PBMCs were isolated by density gradient centrifugation using UniSep ™ blood separation tubes (Accurate Chemical &Scientific; Westbury, NY). The target cells in 50 pL of assay medium (RPMI-1640 with 1% BSA and 100 units / ml of penicillin and streptomycin) were seeded in a 96-well round bottom plate, with 4 x 10 ⁴ / well. Serial dilutions of the test and the control antibody (50 pL / well) were added to plates containing the target cells, followed by incubation at 37 ^S C with 5% carbon dioxide for 30 minutes to allow opsonization. Final antibody concentrations ranged from 0.0051 to 10,000 ng / mL after 5-fold serial dilutions for a total of 10 data points. After incubation, 1.0 x 10 ⁶ PBMC of effector cells in 100 pL of test medium were added to each well to give a 25: 1 ratio of effector: target cells and plates were incubated for an additional 4 hours. The plates were centrifuged at the end of the incubation and the supernatants were tested for lactate dehydrogenase (LDH) activity using a Cytotoxicity Detection ™ Kit (Roche Applied Scinece;
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Indianapolis, IN). The LDH reaction mixture was added to the supernatants and the plates were incubated at room temperature for 15 minutes with constant shaking. The reaction was terminated with 1 Μ H ₃ PO ₄ and the absorbance was measured at 490 nm (the bottom, measured at 650 nm, was subtracted for each well), using a SpectraMax Plus microplate reader. The absorbance of the wells containing only the target cells served as a control for the bottom (low control), while the wells containing target cells dissolved with Triton-X 100 provided the maximum available signal (high control). Antibody-independent cellular cytotoxin (AICC) was measured in wells containing target and effector cells without the addition of antibodies. The specific ADCC extension was calculated as follows:
A490 (Sample) - A490 (AICC)% ADCC - 100 x ------------------------------- A490 (High Control) · Α ₄₉₀ (Low Control) [00279] The ADCC values of the sample dilutions were plotted against the antibody concentration and the dose response curves were adapted to a four-parameter model using SoftMax Pro.
[00280] In a first set of experiments, the ADCC activity of several anti-human TfR constructs was assessed using both a human erythroleukemia cell line (HEL cells) and primary human bone marrow mononuclear cells. The competent anti-human 15G11 TfR1 antibody with bivalent lgG1 effector function and a bispecific form of this antibody with the same anti-BACE1 arm used in the previous examples in a human lgG1 format with D265A and N297G mutations nullifying the effector function (see Example 6) were tested at various concentrations in the ADCC assay, with the use of anti-gD IgG1 WT as a negative control and murine anti-human HLA (class I) as a control
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170/171 positive. The results are shown in Figures 13A and 13B. Either with HEL cells as targets (Fig. 13A) or with bone marrow mononuclear cells as targets (Fig. 13B), monospecific anti-human 15G11 TfR antibody achieved significant ADCC activity. This activity was similar to that of positive control anti-human HLA antibodies on HEL cells and at a robust level even lower than positive control on bone marrow mononuclear cells. The somewhat lower level observed in the bone marrow mononuclear cell experiment is probably due to the fact that only a portion of the heterogeneous mixture of myeloid and erythroid PBMC cells used in the experiment express high levels of TfR, whereas HEL cells show a high level of TfR. consistently high TfR expression across the clonal cell population. In sharp contrast, the anti-human TIR / BACE1 antibody without bispecific effector showed no ADCC activity in either HEL cells or bone marrow mononuclear cells, similar to the negative control.
[00281] In a second set of experiments, the impact of switching the antibody isotype on this assay system was assessed. The procedure of the ADCC assay was identical to that described above, except that all target cells were HEL cells and the effector cells were PMBCs from healthy human donors, or carrying the heterozygous FcyRllla-V / F158 genotype or the FcyRllla-V / genotype. Homozygous V158. All anti-human TfRs tested were bispecific with anti-gD, with three different Ig main chains: wild-type human IgG1, human IgGl with the N297G mutation and human IgG4. An anti-Abeta antibody with human IgG4 main chain was also tested and an anti-human rat HLA (class I) served as a positive control. The results are shown in Figures 14A and 14B. As anticipated, based on the known association between effector cell activation and the V / V 158 genotype (Bowles and Weiner
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2005), ADCC activity was more robustly obtained by donor V / V 158 of PBMCs (approximately 45% of impacted target cells) relative to F / V donors (approximately 25% of impacted target cells) (compare Fig. 14A with Fig. 14B). Anti-TfR / gD with wild-type lgG1 induced robust ADCC in HEL cells, while anti-TfR / gD with lgG1 without effector showed no ADCC activity in HEL cells, replicating the results from the first set of experiments . Notably, at concentrations of 100 ng / mL or greater, anti-TfR / gD of the lgG4 isotype demonstrated mild ADCC activity. This activity was not seen in the anti-Abeta IgG4 results, indicating that TfR binding was necessary for ADCC activity. This finding is correlated with the previous report that lgG4 has a minimal, but measurable, effective function (Adolffson et al., J. Neuroses'. 32 (28): 9677-9689 (2012); van der Zee et al. Clin Exp. Immunol 64: 415-422 (1986)); Tao et al., J. Exp. Med. 173: 1025-1028 (1991)).
[00282] Thus, the findings incorporated in the present application that the depletion of cells of erythroid lineage in mice occur in a TfR-dependent manner and in an effective function, being directly translatable to the human system. Although the aforementioned invention has been described in some detail by way of illustration and examples for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited in this application are expressly incorporated in their entirety as a reference.

权利要求:
Claims (55)
[1]
Referrals
1. USE OF AN ANTIBODY, which binds to a transferrin receptor (TfR), characterized by being to transport a compound through the blood-brain barrier, in which the antibody is coupled to the compound, and in which the antibody binds to TfR with low affinity and in which one or more properties of the antibody have been modified to reduce the impact of the antibody on reticulocyte levels and / or to reduce the severity or presence of acute clinical symptoms in the subject, such as reducing levels of red blood cells in the subject upon administration is decreased or eliminated.
[2]
2. USE, according to claim 1, characterized in that it is for treating a neurological disorder in a subject.
[3]
3. USE, according to claim 2, characterized by the neurological dysfunction being selected from the group consisting of Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), sclerosis amyotrophic lateral (ALS), cystic fibrosis, Angelman syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer and traumatic brain injury.
[4]
USE, according to one of claims 1 to 3, characterized in that the subject is a human.
[5]
5. USE, according to one of claims 2 to 4, characterized in that one or more properties are selected from the effector function of the antibody Fc region and the complement complement activation function of the antibody, and in which the effector function or function of complement activation was reduced or eliminated compared to a wild-type antibody of the same isotype.
[6]
6. USE, according to claim 5, characterized by the effector function being reduced or eliminated by a method selected from
Petition 870160021356, of 05/19/2016, p. 36/217 reduction of antibody glycosylation, modification of the antibody isotype to an isotype that naturally has reduced or eliminated effector function, and modification of the Fc region.
[7]
7. USE, according to claim 6, characterized in that the glycosylation of the antibody is reduced by a method selected from: production of the antibody in an environment that does not allow glycosylation of the wild type, removal of carbohydrate groups already present in the antibody, and modifying the antibody so that wild-type glycosylation does not occur.
[8]
USE, according to claim 7, characterized in that the antibody is produced in a non-mammalian cell production system, or where the antibody is produced synthetically.
[9]
Use according to one of claims 1 to 8, characterized in that the Fc region of the antibody comprises a mutation at position 297, so that the asparagine residue of the wild type at that position is replaced by another amino acid that interferes with glycosylation at that position .
[10]
10. USE, according to claim 6, characterized in that the effector function or complement activation function is reduced or eliminated by deleting all or part of the Fc region, or by making the antibody so as not to include an Fc region or non Fc region competent for effector function or complement activation function, or where the modification is selected from: a point mutation in the Fc region to impair the connection to one or more Fc receptors selected from the following positions: 238 , 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301,303, 322, 324, 327, 329, 333 , 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438 and 439; a point mutation in the Fc region to impair binding to Clq
Petition 870160021356, of 05/19/2016, p. 37/217 selected from the following positions: 270, 322, 329 and 321; elimination of some or all of the Fc region, and a point mutation at position 132 of the CH1 domain.
[11]
11. USE, according to one of claims 1 to 10, characterized in that the antibody coupled to the compound, is administered with an additional compound selected from erythropoietin (EPO), a supplement of iron, vitamin C, folic acid and vitamin B12, or wherein the additional compound is red blood cells or reticulocytes from the same or another subject.
[12]
12. USE, according to one of claims 1 to 11, characterized in that the subject is monitored for red blood cell depletion.
[13]
13. USE, according to one of claims 1 to 12, characterized in that the compound is a drug for neurological dysfunction or an imaging agent.
[14]
14. USE, according to one of claims 1 to 13, characterized in that the antibody does not impact the binding of TfR to transferrin.
[15]
15. USE, according to one of claims 1 to 14, characterized in that the antibody has an IC50 for TfR of about 1 nM to about 100 μΜ, or about 5 nM to about 100 μΜ, or about 50 nM to about 100 μΜ, or from about 100 nM to about 100 μΜ.
[16]
16. USE, according to one of claims 1 to 15, characterized in that the antibody has an affinity for TfR of about 5 nM to about 50 μΜ.
[17]
17. USE, according to one of claims 1 to 16, characterized in that the antibody coupled to the compound has an affinity for TfR of about 30 nM to about 30 μΜ, or from about 30 nM to about 1 μΜ.
[18]
18. USE, according to one of claims 1 to 17,
Petition 870160021356, of 05/19/2016, p. 38/217
4/10 characterized by the antibody coupled to the compound, having a dissociation half life for TfR of about 30 seconds to about 5 minutes, or about 30 seconds to about 2 minutes.
[19]
19. USE according to one of claims 1 to 18, characterized in that the antibody is a multispecific antibody and the compound optionally forms a portion of the multispecific antibody.
[20]
20. USE, according to claim 19, characterized in that the multispecific antibody comprises a first antigen binding site that binds TfR and a second antigen binding site that binds a brain antigen.
[21]
21. USE, according to claim 20, characterized in that the cerebral antigen is selected from the group consisting of: beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), epidermal growth factor receptor 2 human (HER2), tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine 2 repeat rich kinase (LRRK2), parquine, presenilin 1, presenilin 2, gamma secretase, receptor 6 of death (DR6), amyloid precursor protein (APP), neurotrophin p75 receptor (p75NTR) and caspase 6.
[22]
22. USE, according to claim 20, characterized in that the multispecific antibody binds to both TfR and BACE1 or, wherein the multispecific antibody binds to both TfR and Abeta.
[23]
23. METHOD FOR MAKING AN ANTIBODY useful for transporting a compound through BBB with enhanced safety, characterized by understanding to select a specific antibody for a transferrin receptor (TfR) that has low affinity for TfR, and to modify one or more properties of the antibody to reduce the impact of the antibody on reticulocyte levels and / or reduce the severity or presence of acute clinical symptoms in a subject, such as reduced levels of
Petition 870160021356, of 05/19/2016, p. 39/217 red blood cells in a subject, upon administration of the antibody, is decreased or eliminated, compared to the unmodified antibody.
[24]
24. METHOD, according to claim 23, characterized in that one or more properties are selected from the effector function of the antibody Fc region and the complement complement activation function of the antibody, and in which, the effector function or the function of complement activation was reduced or eliminated compared to a wild-type antibody of the same isotype.
[25]
25. METHOD according to claim 24, characterized in that the effector function is reduced or eliminated by a method selected from the reduction of antibody glycosylation, modification of the antibody isotype to an isotype that naturally has reduced or eliminated effector function, and modification of the Fc region.
[26]
26. METHOD, according to claim 25, characterized in that the glycosylation of the antibody is reduced by a method selected from: production of the antibody in an environment that does not allow wild-type glycosylation, removal of carbohydrate groups already present in the antibody, and modification of the antibody so that wild-type glycosylation does not occur.
[27]
27. METHOD according to claim 26, characterized in that the antibody is produced in a non-mammalian cell production system, or where the antibody is produced synthetically.
[28]
28. METHOD according to claim 26, characterized in that the FC region of the antibody comprises a mutation at position 297, so that the wild-type asparagine residue at that position is replaced by another amino acid that interferes with glycosylation at that position.
[29]
29. METHOD, according to claim 24, characterized
Petition 870160021356, of 05/19/2016, p. 40/217 because the effector function or the complement activation function is reduced or eliminated by the deletion of all or part of the Fc region, or by the elaboration of the antibody so as not to include a competent Fc region for the effector function or activation function of complement, or the modification is selected from: a point mutation in the Fc region to impair binding to one or more Fc receptors selected from the following positions: 238, 239, 248, 249, 252, 254, 265, 268 , 269, 270, 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301,303, 322, 324, 327, 329, 333, 338, 340, 373, 376, 382, 388, 389 , 414, 416, 419, 434, 435, 437, 438 and 439; a point mutation in the Fc region to impair the connection to 01 q selected from the following positions: 270, 322, 329 and 321; elimination of some or all of the Fc region, and a point mutation at position 132 of the CH1 domain.
[30]
30. METHOD according to one of claims 23 to 29, characterized in that the antibody has an IC50 for TfR of about 1 nM to about 100 μΜ, or about 5 nM to about 100 μΜ, or about 50 nM to about 100 μΜ, or from about 100 nM to about 100 μΜ.
[31]
31. METHOD according to one of claims 23 to 30, characterized in that the antibody is coupled with a therapeutic compound.
[32]
32. METHOD according to claim 31, characterized in that the therapeutic compound is a drug for neurological dysfunction.
[33]
33. METHOD according to one of claims 23 to 30, characterized in that the antibody is a multispecific antibody comprising a first antigen binding site that binds TfR and a second antigen binding site that binds a brain antigen.
[34]
34. METHOD, according to claim 33, characterized by the cerebral antigen being selected from the group consisting of: beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), epidermal growth factor receptor 2 human (HER2), tau,
Petition 870160021356, of 05/19/2016, p. 41/217
7/10 apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), kinase rich in repeat leucine 2 (LRRK2), parquine, presenilin 1, presenilin 2, gamma secretase, receptor 6 of death (DR6 ), amyloid precursor protein (APP), neurotrophin p75 receptor (p75NTR) and caspase 6.
[35]
35. METHOD according to claim 33, characterized by the multispecific antibody binding to both TfR and BACE1, or (ii) the multispecific antibody binding to both TfR and Abeta.
[36]
36. METHOD according to one of claims 23 to 35, characterized in that the antibody has an affinity for TfR of about 5 nM to about 50 μΜ.
[37]
37. METHOD according to one of claims 23 to 36, characterized in that the antibody has a dissociation half life for TfR of about 30 seconds to about 5 minutes, or about 30 seconds to about 2 minutes.
[38]
38. METHOD according to one of claims 23 to 37, characterized in that the antibody does not inhibit the binding of TfR to transferrin.
[39]
39. ANTIBODY, characterized by binding to a TfR, where the antibody affinity for TfR is about 5 nM to about 50 μΜ or the dissociation half life of the antibody to TfR is about 30 seconds at about 2 minutes, and in which one or more properties of the antibody has been modified to reduce the impact of the antibody on reticulocyte levels and / or to reduce the severity or presence of acute clinical symptoms.
[40]
40. ANTIBODY, according to claim 39, characterized in that one or more properties is selected from the effector function of the antibody Fc region and the complement complement activation function of the antibody, and in which the effector function or activation function of complement has been reduced or eliminated in relation to a wild-type antibody
Petition 870160021356, of 05/19/2016, p. 42/217 of the same isotype.
[41]
41. ANTIBODY, according to claim 40, characterized in that the effector function is reduced or eliminated by a method selected from the production of the antibody in an environment that does not allow wild-type glycosylation; removing carbohydrate groups already present in the antibody, and modifying the antibody so that wild-type glycosylation does not occur.
[42]
42. ANTIBODY according to claim 41, characterized in that the antibody is produced in a non-mammalian cell production system, or where the antibody is produced synthetically.
[43]
43. ANTIBODY according to claim 42, characterized in that the Fc region of the antibody comprises a mutation at position 297, so that the wild-type asparagine residue at that position is replaced by another amino acid that interferes with glycosylation at that position.
[44]
44. ANTIBODY according to claim 40, characterized in that the effector function or the complementary activation function is reduced or eliminated by deleting all or part of the Fc region, or by making the antibody so as not to include a competent Fc region for effector function, or where the modification is selected from: a point mutation of the Fc region to impair the connection to one or more Fc receptors selected from the following positions: 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301,303, 322, 324, 327, 329, 333, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438 and 439; a point mutation in the Fc region to impair binding to C1q selected from the following positions: 270, 322, 329 and 321; elimination of some or all of the Fc region, and a point mutation at position 132 of the CH1 domain.
Petition 870160021356, of 05/19/2016, p. 43/217
[45]
45. ANTIBODY according to one of claims 39 to
44, characterized in that the antibody is coupled with a therapeutic compound.
[46]
46. ANTIBODY according to claim 45, characterized in that the therapeutic compound is a drug for neurological dysfunction.
[47]
47. ANTIBODY according to claim 45, characterized in that the antibody is a multispecific antibody and the compound optionally forms a portion of the multispecific antibody.
[48]
48. ANTIBODY according to claim 47, characterized in that the antibody is a multispecific antibody comprising a first antigen binding site that binds TfR and a second antigen binding site that binds a brain antigen.
[49]
49. ANTIBODY according to claim 48, characterized in that the cerebral antigen is selected from the group consisting of: beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), receptor 2 for human epidermal growth (HER2), tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine 2 repeat rich kinase (LRRK2), parchin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR) and caspase 6.
[50]
50. ANTIBODY according to claim 49, characterized in that the multispecific antibody binds both TfR and BACE1, or in which the multispecific antibody binds both TfR and Abeta.
[51]
51. ANTIBODY according to one of claims 39 to
50, characterized in that the antibody does not inhibit TfR binding to transferrin.
[52]
52. USE OF AN ANTIBODY that binds with low affinity to a TfR, as defined in one of claims 1 to 51, characterized
Petition 870160021356, of 05/19/2016, p. 44/217
10/10 for the manufacture of a drug to treat a neurological disorder, in which one or more properties of the antibody has been modified to reduce the impact of the antibody on reticulocyte levels and / or to reduce the severity or presence of acute clinical symptoms .
[53]
53. USE OF AN ANTIBODY, as defined in one of claims 39 to 51, characterized in that it is for the manufacture of a medicine to treat a neurological dysfunction.
[54]
54. ANTIBODY, which binds with low affinity to a TfR, characterized by being for use in the treatment of neurological dysfunction, in which one or more properties of the antibody has been modified to reduce the impact of the antibody on reticulocyte and / or levels reduce the severity or presence of acute clinical symptoms.
[55]
55. ANTIBODY according to one of claims 39 to
51, characterized for being for use in the treatment of a neurological disorder.

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US20210395381A1|2018-09-14|2021-12-23|Ossianix, Inc.|Tfr-specific binding moieties and transcytosis method to select vnars that cross cellular barriers|

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EP3903816A1|2018-12-28|2021-11-03|Kyowa Kirin Co., Ltd.|Bispecific antibody binding to tfr|

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CN113474369A|2019-01-09|2021-10-01|维克塔-霍洛斯公司|Transferrin receptor binding molecules, conjugates thereof, and uses thereof|

法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-06| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-03-13| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-03-20| B06I| Publication of requirement cancelled [chapter 6.9 patent gazette]|Free format text: ANULADA A PUBLICACAO CODIGO 6.6.1 NA RPI NO 2462 DE 13/03/2018 POR TER SIDO INDEVIDA. |

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2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: C07K 16/28 (2006.01), C07K 16/40 (2006.01), C07K 1 |

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2020-06-23| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-10-05| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-10-19| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2022-01-11| B09B| Patent application refused [chapter 9.2 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201261649878P| true| 2012-05-21|2012-05-21|

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US201261698495P| true| 2012-09-07|2012-09-07|

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US201361763915P| true| 2013-02-12|2013-02-12|

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PCT/US2013/041860|WO2013177062A2|2012-05-21|2013-05-20|Methods for improving safety of blood-brain barrier transport|

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