HK1215584B - Anti-transferrin receptor antibodies and methods of use - Google Patents

Description

Transferrin receptor antibodies and methods of use thereof

Field of the Invention

The present invention relates to antibodies to transferrin receptor and methods of using the same.

Related applications

This application claims the benefit of U.S. Provisional Application No. 61/825,477, filed May 20, 2013, which is incorporated herein by reference in its entirety.

Sequence Listing

The sequence listing is submitted with this specification in an ASCII format text file via EFS-Web, with the file name "P5641R1-WO_SL.txt" and a size of 154,599 bytes, on May 16, 2014. The sequence listing submitted via EFS-Web is part of this specification and is hereby fully incorporated herein by reference.

background

Brain penetration of macromolecular drugs is severely limited by the largely impermeable blood-brain barrier (BBB). Among the various strategies to overcome this obstacle, there is a transcytosis transport pathway that utilizes endogenous receptors expressed in the brain capillary endothelium. Recombinant proteins such as monoclonal antibodies against these receptors have been designed to allow receptor-mediated delivery of macromolecules to the brain. Strategies have been proposed to maximize brain uptake while minimizing reverse transcytosis back into the blood and also maximizing the degree of accumulation after treatment administration, which has been addressed by the discovery that antibodies with low BBB receptor affinity provide substantial increases in the potential for BBB transport and CNS retention of the associated therapeutic moiety/molecule relative to typical high-affinity antibodies for such receptors (Atwal et al., Sci. Transl. Med. 3, 84ra43 (2011); Yu et al., Sci. Transl. Med. 25 May 2011: Vol. 3, Issue 84, p. 84ra44). However, those antibodies did not specifically bind to human and primate TfR.

Overview

Monoclonal antibodies have great therapeutic potential for treating neurological or central nervous system (CNS) diseases, but the blood-brain barrier (BBB) restricts their access to the brain. Past studies have shown that a very small percentage (about 0.1%) of IgG circulating in the bloodstream crosses the BBB and enters the CNS (Felgenhauer, Klin. Wschr. 52: 1158-1164 (1974)), where the CNS concentration of the antibody may not be sufficient to allow a robust effect. It has been previously discovered that the percentage of antibodies distributed into the CNS can be increased by exploiting BBB receptors (i.e., transferrin receptor, insulin receptor, etc.) (see, for example, WO9502421). For example, anti-BBB receptor antibodies can be made multispecific to target one or more desired antigens in the CNS, or one or more heterologous molecules can be conjugated to the anti-BBB receptor antibodies; in either case, the anti-BBB receptor antibodies can assist in the delivery of therapeutic molecules across the BBB into the CNS.

However, targeting BBB receptors with traditional specific high-affinity antibodies generally causes a limited increase in BBB transport. The applicant later found that in the anti-BBB antibodies studied, the number of antibodies taken in and distributed in the CNS was inversely correlated with their binding affinity to BBB receptors. For example, relative to the anti-TfR antibodies with higher affinity, antibodies with low affinity for transferrin receptor (TfR) administered at therapeutic dose levels significantly increased the BBB transport and CNS retention of anti-TfR antibodies, and therapeutic concentrations can be more easily obtained in the CNS (Atwal et al., Sci. Transl. Med. 3, 84ra43 (2011)). Evidence of this BBB transport was obtained using bispecific antibodies, which bind to both TfR and amyloid precursor protein (APP) cleavage enzyme β-secretase (BACE1). Compared to monospecific anti-BACE1 alone, a single systemic administration of a bispecific anti-TfR/BACE1 antibody engineered using the methods of the present invention not only resulted in significant antibody uptake in the brain, but also significantly reduced brain Aβ _1-40 levels, suggesting that BBB permeability influences anti-BACE1 efficacy (Atwal et al., Sci. Transl. Med. 3, 84ra43 (2011); Yu et al., Sci. Transl. Med. 3, 84ra44 (2011)).

Those data and experiments have highlighted that the method for using lower affinity antibodies increases the several causal mechanisms that antibodies are taken into CNS.First, high-affinity anti-BBB receptor (BBB-R) antibodies (for example, anti- ^TfRA , it is from Atwal et al. and Yu et al., as previously described) limit brain uptake by rapidly saturating the BBB-R in the cerebral vascular system, therefore reducing the total amount of the antibody taken into the brain and also limiting its distribution to the vascular system. Surprisingly, reducing the affinity for BBB-R improves brain uptake and distribution, and observing the obvious movement of the positioning of the neurons and related neuropil distributed in the CNS from the vascular system. Second, it is proposed that the lower affinity of antibodies to BBB-R has weakened the ability of antibodies to return to the vascular side of the membrane from the CNS side of the BBB by BBB-R, because the overall affinity of antibodies to BBB-R is low and due to the rapid diffusion of antibodies into the CNS compartment (compartment), the local concentration of antibodies at the CNS side of the BBB is unsaturated. Third, in vivo, and as observed for the TfR system, antibodies with lower affinity for BBB-R are not effectively cleared from the system like those with higher affinity for BBB-R, and are therefore maintained at higher circulating concentrations compared to their higher affinity counterparts. This is advantageous because circulating antibody levels for lower affinity antibodies are maintained at therapeutic levels for longer periods than for higher affinity antibodies, which therefore improves brain uptake of antibodies over a longer period of time. In addition, this increase in plasma and brain exposure can reduce the frequency of clinical medication, which will have potential benefits, not only for patient compliance and convenience, but also in alleviating any potential side effects or off-target effects of the antibody and/or the therapeutic compounds coupled thereto.

The low affinity BBB-R antibodies described in the work cited above were selected/modified to avoid interfering with the natural binding between transferrin and TfR and thus to avoid potential iron transport-related side effects. However, when some of these antibodies were administered in mice, some significant side effects were observed. The mice showed a primary reaction of significant depletion of the reticulocyte population, accompanied by acute clinical symptoms of rapid onset. Although the mice recovered promptly from both the acute clinical symptoms and the reduced reticulocyte levels, it is clear that for anti-TfR antibodies that can be safely used as therapeutic molecules, this effect on reticulocytes needs to be avoided or mitigated. It was found that the main reaction to anti-TfR administration (significant reticulocyte depletion and acute clinical signs) was largely driven by the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the antibody, while the remaining reticulocyte depletion effect was mediated by the complement pathway.

These previous studies utilized mouse antibodies that specifically bind to mouse TfR but do not specifically recognize primate or human TfR. Therefore, the present invention provides antibodies and functional portions thereof that do specifically recognize both primate and human TfR to facilitate safety and efficacy studies using the antibodies in primates before therapeutic or diagnostic applications in humans. In vitro studies using human erythroblast lines and primary bone marrow cells treated with the anti-human TfR antibodies of the present invention demonstrated that strong TfR-positive erythrocyte depletion as in mice can also be observed in human/primate cell systems (e.g., see Example 4). Therefore, the present invention also provides modifications to the antibodies of the present invention to greatly reduce or eliminate the unnecessary reduction of TfR-expressing reticulocyte populations when anti-TfR is administered, while still allowing the enhanced BBB transport, increased CNS distribution, and CNS retention provided by anti-human/primate TfR antibodies administered at therapeutic concentrations. The present invention provides several general methods for mitigating the observed effects of the anti-TfR antibodies of the present invention on primary and residual reticulocyte depletion, and can be used alone or in combination.

In one approach, the effector function of the anti-human/cynomolgus monkey (cyno) TfR antibody is reduced or eliminated so as to reduce or eliminate ADCC activity. In another approach, the affinity of the anti-human/cynomolgus monkey TfR antibody to human or primate TfR is further reduced so that the interaction of the antibody with the reticulocyte population is less damaging to the population. A third approach involves reducing the amount of anti-human/cynomolgus monkey TfR antibody present in the plasma to reduce the exposure of the reticulocyte population to potentially harmful concentrations of the antibody. A fourth approach seeks to protect, stabilize and/or supplement the reticulocyte population so as to avoid, reduce or mitigate any potential depletion of the reticulocyte population in the circulation or bone marrow caused by the administration of the anti-human/cynomolgus monkey TfR antibody.

As described herein, reduction or elimination of effector function can be achieved by: (i) reducing or eliminating wild-type mammalian glycosylation of the antibody (e.g., by producing the antibody in an environment in which such glycosylation cannot occur, by mutating one or more carbohydrate attachment sites so that the antibody cannot be glycosylated, or by chemically or enzymatically removing one or more carbohydrates from the antibody after it has been glycosylated); (ii) by reducing or eliminating the Fc receptor binding ability of the anti-human/cynomolgus monkey TfR antibody (e.g., by mutation of the Fc region, deletion within the Fc region, or elimination of the Fc region); or (iii) by utilizing an antibody isotype known to have minimal or no effector function (i.e., including but not limited to IgG4).

As described herein, reducing antibody complement activation can be achieved by reducing or eliminating the C1q binding ability of the anti-human/cynomolgus monkey TfR antibody (e.g., by mutation of the Fc region, deletion within the Fc region, or elimination of the Fc region, or by modifying the non-Fc portion of the anti-human/cynomolgus monkey TfR antibody), or by otherwise inhibiting the activation or activity of the complement system (e.g., by co-administering one or more complement pathway activation or complement pathway activity inhibitors).

When binding of an anti-human/cynomolgus TfR antibody to human or cynomolgus TfR on reticulocytes or other cell types expressing high levels of TfR induces their depletion, as with the anti-human/cynomolgus TfR antibodies exemplified herein, reducing the binding of the antibody to human or cynomolgus TfR on reticulocytes or other cell types should in turn reduce the amount of reticulocyte or other cell type depletion observed in the circulation or bone marrow upon antibody administration. Any of the methods described herein and as shown in the Examples can be used to modify the affinity of an anti-human/cynomolgus TfR antibody for primate or human TfR.

Reducing the amount of anti-human/cynomolgus monkey TfR antibodies present in the plasma in order to reduce the exposure of the reticulocyte population to potentially harmful concentrations of antibodies can be achieved in a variety of ways. One approach is to simply reduce the amount of antibody administered, potentially while also increasing the frequency of administration, so as to reduce the maximum concentration in the plasma, but maintain serum levels sufficient for efficacy while still below the threshold for cell-consuming side effects. Another approach that can be combined with dosing improvements is to select or engineer an anti-TfR antibody with pH-sensitive binding to TfR so that it binds to cell surface TfR in plasma at pH 7.4 with the desired low affinity as described herein, but upon internalization to the endosomal compartment, at the relatively lower pH of the compartment (pH 5.5-6.0), this binding to TfR is rapidly and significantly reduced. This dissociation can protect the antibody from antigen-mediated clearance or increase the amount of antibody delivered to the CNS or recovered across the BBB - in either case, the effective concentration of the antibody is increased relative to anti-TfR antibodies that do not contain this pH sensitivity, without increasing the administered dose of the antibody, and in turn potentially allowing lower antibody doses with a lower risk of side effects.

Protection, stabilization and/or replenishment of the reticulocyte population can be achieved using pharmaceutical or physical methods. In addition to the anti-human/cynomolgus monkey TfR antibody, at least one additional therapeutic agent that mitigates the adverse side effects of the antibody on the reticulocyte population can be co-administered (simultaneously or sequentially). Examples of such therapeutic agents include, but are not limited to, erythropoietin (EPO), iron supplements, vitamin C, folic acid, and vitamin B12. Physical replacement of red blood cells (i.e., reticulocytes) can also be performed, for example, by transfusion with similar cells that can be from another individual with a similar blood type or that have been previously extracted from a subject to whom the anti-human/cynomolgus monkey TfR antibody is administered.

One of ordinary skill in the art will appreciate that any combination of the foregoing approaches can be employed to engineer an antibody (and/or its dosage regimen) to achieve an optimal balance between: (i) the desired low affinity for primate or human TfR that will maximize transport of the antibody and any conjugated compound into the CNS; (ii) the affinity of the conjugated compound for its CNS antigen (including, as a non-limiting example, a second or additional antigen binding specificity in an anti-human/cynomolgus monkey TfR antibody), as this relates to the amount of compound that needs to be present in the CNS to have a therapeutic effect; (iii) the clearance rate of the anti-human/cynomolgus monkey TfR antibody; (iv) instability of the anti-TfR/conjugated compound at low pH to promote release of the conjugated compound on the CNS/brain side of the BBB, and (v) effects on the reticulocyte population.

It will also be understood that the reticulocyte depletion effect recognized herein of anti-TfR antibody administration can be used to treat any disease or condition in which excessive proliferation of reticulocytes is a problem. For example, in congenital polycythemia or neoplastic polycythemia vera, elevated red blood cell counts due to, for example, excessive proliferation of reticulocytes lead to blood thickening and accompanying physiological symptoms. Administration of the anti-human/cynomolgus monkey TfR antibodies of the present invention, in which at least some effector function of the antibody is retained, will allow selective removal of immature reticulocyte populations without affecting normal transferrin transport to the CNS. As is well known in the art, the administration of such antibodies can be adjusted so that acute clinical symptoms can be minimized (i.e., at very low doses or at wide intervals).

Both anti-TfR/BACE1 and anti-TfR/Aβ are promising and novel candidate therapeutic agents for the treatment of Alzheimer's disease. In addition, bispecific targeting technology based on receptor-mediated transport (RMT) has opened the door to a wide range of potential therapeutic agents for CNS diseases. The present invention provides a method for engineering BBB-permeable therapeutic agents that greatly improves trans-BBB transport and CNS distribution of therapeutic agents without depletion of reticulocytes.

Thus, in a first embodiment, the invention provides an isolated antibody that binds to human transferrin autoreceptor (TfR) and primate TfR, wherein the antibody does not inhibit the binding of transferrin to TfR. In one aspect, the binding is specific binding. In another aspect, the antibody also does not inhibit the binding of human hemochromatin ("HFE") to TfR. In one aspect, the binding is specific binding. In one aspect, the antibody is a monoclonal antibody. In another aspect, the antibody is a human antibody. In another aspect, the antibody is a humanized antibody. In another aspect, the antibody is a chimeric antibody. In another aspect, the antibody is an antibody fragment that binds to human TfR and primate TfR. In another aspect, the primate TfR is from a cynomolgous monkey.

In one aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 31, 33, and 34, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 36, 38, and 39, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 36, 40, and 34, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 42, 43, and 44, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 31, 33, and 34, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 46, 48 and 49, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 52, 54 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 52, 58 and 59, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 62, 63 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 52, 65 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 68, 69 and 70, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 73, 75 and 76, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 79, 81 and 82, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 79, 83 and 84, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 87, 89 and 90, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 93, 95 and 96, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 99, 101 and 102, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 127, 33 and 34, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 52, 156 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 52, 157 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 155, 54 and 55, respectively.

In one aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 37, 38, and 39, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 32, 40, and 34, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 37, 43, and 44, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 47, 48 and 49, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 54 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 58 and 59, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 63 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 65 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 69 and 70, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 74, 75 and 76, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 80, 81 and 82, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 80, 83 and 84, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 88, 89 and 90, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 94, 95 and 96, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 100, 101 and 102, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 156 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 157 and 55, respectively.

In one aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 29, 30, and 31, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 35, 30, and 36, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 41, 30, and 42, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 29, 30, and 31, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 45, 30, and 46, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 50, 51, and 52, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 56, 57, and 52, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 60, 61, and 62, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 60, 64, and 52, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 66, 67, and 68, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 71, 72, and 73, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 77, 78, and 79, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 85, 86, and 87, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 91, 92, and 93, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 97, 98, and 99, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 29, 30, and 127, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 50, 51, and 155, respectively.

In one aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 29, 30, and 31, respectively, and HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 35, 30, and 36, respectively, and HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 37, 38, and 39, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 35, 30 and 36, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 32, 40 and 34. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 41, 30 and 42, respectively, and HVR-H1, HVR-H2 and HVR-H comprising the amino acid sequences of SEQ ID NOs: 37, 43 and 44, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 29, 30, and 31, respectively, and HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 45, 30, and 46, respectively, and HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 50, 51 and 52, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 54 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 56, 57 and 52, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 58 and 59, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 60, 61 and 62, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 63 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 60, 64 and 52, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 65 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 66, 67 and 68, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 69 and 70, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 71, 72 and 73, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 74, 75 and 76, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 77, 78 and 79, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 80, 81 and 82, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 77, 78 and 79, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 80, 83 and 84, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 85, 86 and 87, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 88, 89 and 90, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 91, 92 and 93, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 94, 95 and 96, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 97, 98, and 99, respectively, and HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 100, 101, and 102, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 29, 30, and 127, respectively, and HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 50, 51 and 52, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 156 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 50, 51 and 52, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 157 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 50, 51 and 155, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 54 and 55, respectively.

In one aspect of the above embodiments, the antibody comprises at least one HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 or HVR-L3 sequence selected from the group consisting of: an HVR-H1 sequence of SEQ ID NO: 47, 53 or 100; an HVR-H2 sequence of SEQ ID NO: 48, 69, 101, 156 or 157; an HVR-H3 sequence of SEQ ID NO: 49, 76 or 102; an HVR-L1 sequence of SEQ ID NO: 45, 66 or 97; an HVR-L2 sequence of SEQ ID NOs: 30, 67 or 98; and an HVR-L3 sequence of SEQ ID NOs: 46, 68 or 102.

In one aspect of the above embodiments, the antibody comprises: (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 7, 8, 9, 10, 15, 16, 17, 18, 20, 25, 26, 27, 28, 108, 114, 120, 126, 153, or 154; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4, 5, 6, 11, 12, 13, 14, 19, 21, 22, 23, 24, 105, 111, 117, 123, or 151; or (c) the VH sequence of (a) and the VL sequence of (b). In one such aspect, the antibody comprises the VL sequence of SEQ ID NO: 4 and the VH sequence of SEQ ID NO: 7. In another such aspect, the antibody comprises the VL sequence of SEQ ID NO: 5 and the VH sequence of SEQ ID NO: 8. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 5 and a VH sequence of SEQ ID NO: 9. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 6 and a VH sequence of SEQ ID NO: 10. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 11 and a VH sequence of SEQ ID NO: 15. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 12 and a VH sequence of SEQ ID NO: 16. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 13 and a VH sequence of SEQ ID NO: 17. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 14 and a VH sequence of SEQ ID NO: 18. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 19 and a VH sequence of SEQ ID NO: 20. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 21 and a VH sequence of SEQ ID NO: 25. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 22 and a VH sequence of SEQ ID NO: 26. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 23 and a VH sequence of SEQ ID NO: 27. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 24 and a VH sequence of SEQ ID NO: 28. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 105 and a VH sequence of SEQ ID NO: 108. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 111 and a VH sequence of SEQ ID NO: 114. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 117 and a VH sequence of SEQ ID NO: 120. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 123 and a VH sequence of SEQ ID NO: 126. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 105 and a VH sequence of SEQ ID NO: 153. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 105 and a VH sequence of SEQ ID NO: 154. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 151 and a VH sequence of SEQ ID NO: 108. In another such aspect, the antibody comprises a VH sequence of (a) SEQ ID NO: 108, (b) SEQ ID NO: 114, (c) SEQ ID NO: 120, or (d) SEQ ID NO: 126. In another such aspect, the antibody comprises a VL sequence of (a) SEQ ID NO: 105, (b) SEQ ID NO: 111, (c) SEQ ID NO: 117, or (d) SEQ ID NO: 123.

In one aspect of the above embodiment, the antibody is selected from the group consisting of antibodies 7A4, 8A2, 15D2, 10D11, 7B10, 15G11, 16G5, 13C3, 16G4, 16F6, 7G7, 4C2, 1B12, and 13D4. In one such aspect, the antibody is 7A4. In another such aspect, the antibody is 8A2. In another such aspect, the antibody is 15D2. In another such aspect, the antibody is 10D11. In another such aspect, the antibody is 7B10. In another such aspect, the antibody is 15G11. In another such aspect, the antibody is 16G5. In another such aspect, the antibody is 13C3. In another such aspect, the antibody is 16G4. In another such aspect, the antibody is 16F6. In another such aspect, the antibody is 7G7. In another such aspect, the antibody is 4C2. In another such aspect, the antibody is 1B12. In another such aspect, the antibody is 13D4.

In one aspect of any of the foregoing, the antibody is further affinity matured. In one such aspect, the antibody is selected from the group consisting of: 15G11.v1, 15G11.v2, 15G11.v3, 15G11.v4, 15G11.v5, 7A4.v1; 7A4.v2, 7A4.v3, 7A4.v4, 7A4.v5, 7A4.v6, 7A4.v7, 7A4.v8, 7A4.v9, 7A4.v10, 7A4.v11, 7A4.v12, 7A4.v13, 7A4.v14, 7A4.v15, 7G7.v1, 16F6.v1, 16F6.v2, 16F6.v3, 16F6.v4, 15G11.N52A, 15G11.T53A, and 15G11.W92A. In one such aspect, the antibody is 15G11.v1. In another such aspect, the antibody is 15G11.v2. In another such aspect, the antibody is 15G11.v3. In another such aspect, the antibody is 15G11.v4. In another such aspect, the antibody is 15G11.v5. In another such aspect, the antibody is 7A4.v1. In another such aspect, the antibody is 7A4.v2. In another such aspect, the antibody is 7A4.v3. In another such aspect, the antibody is 7A4.v4. In another such aspect, the antibody is 7A4.v5. In another such aspect, the antibody is 7A4.v6. In another such aspect, the antibody is 7A4.v7. In another such aspect, the antibody is 7A4.v8. In another such aspect, the antibody is 7A4.v9. In another such aspect, the antibody is 7A4.v10. In another such aspect, the antibody is 7A4.v11. In another such aspect, the antibody is 7A4.v12. In another such aspect, the antibody is 7A4.v13. In another such aspect, the antibody is 7A4.v14. In another such aspect, the antibody is 7A4.v15. In another such aspect, the antibody is 7G7.v1. In another such aspect, the antibody is 16F6.v1. In another such aspect, the antibody is 16F6.v2. In another such aspect, the antibody is 16F6.v3. In another such aspect, the antibody is 16F6.v4. In another such aspect, the antibody is 15G11.N52A. In another such aspect, the antibody is 15G11.T53A. In another such aspect, the antibody is 15G11.W92A.

In one aspect of the above embodiment, the antibody is modified at one or more amino acid positions of VH or VL to the amino acids shown in Figures 4E-1 and 4E-2 for the positions. In another aspect of the above embodiment, the antibody comprises a sequence corresponding to the sequence described for any one of the clones in Figures 3 and 4 and Figures 4E-1 and 4E-2 or one or more of these sequences or one or more HVR sequences. In another aspect of the above embodiment, the antibody comprises a VH or VL sequence corresponding to the VH or VL sequence described for any one of the clones in Figures 3 and 4 and Figures 4E-1 and 4E-2. In another aspect of the above embodiment, the antibody comprises one or more HVR sequences corresponding to one or more of those described for any one of the clones in Figures 3 and 4 and Figures 4E-1 and 4E-2.

In one aspect of the above embodiment, the antibody is coupled to a therapeutic compound. In another aspect of the above embodiment, the antibody is coupled to an imaging agent or label. In one such aspect, the antibody is a multispecific antibody, and the therapeutic compound optionally forms part of the multispecific antibody. In one such aspect, the multispecific antibody comprises a first antigen binding site that binds to TfR and a second antigen binding site that binds to a brain antigen. In one such aspect, the brain antigen is selected from the group consisting of: beta-secretase 1 (BACE1), Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E (ApoE), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR) and caspase 6. In another such aspect, the multispecific antibody binds to both TfR and BACE1. In another such aspect, the multispecific antibody binds to both TfR and Aβ. In another such aspect, the therapeutic compound is a neurological disease drug.

In one aspect of the above embodiments, the present invention provides isolated nucleic acids encoding any of the aforementioned antibodies. In another aspect, the present invention provides host cells comprising the nucleic acids. In another aspect, the present invention provides methods for preparing any of the aforementioned antibodies, the methods comprising culturing the host cells to produce the antibodies, and optionally further comprising recovering the antibodies from the host cells.

In one aspect of the above embodiments, the invention provides a pharmaceutical formulation comprising any of the aforementioned antibodies and a pharmaceutically acceptable carrier.

In one aspect of the above-mentioned embodiment, the present invention provides any of the aforementioned antibodies for use as a medicine. In another aspect of the above-mentioned embodiment, the present invention provides the use of any of the aforementioned antibodies in the preparation of a medicament for treating a neurological disease. In one such aspect, the neurological disease is selected from the group consisting of: neuropathy disorder, neurodegenerative disease, cancer, ocular disease disorder, seizure disorder, lysosomal storage disease, amyloidosis, viral or microbial disease, ischemia, behavioral disorder, and CNS inflammation.

In another aspect of the above embodiments, the invention provides any of the aforementioned antibodies for use in treating a neurological disease. In one such aspect, the neurological disease is selected from the group consisting of a neurological disorder, a neurodegenerative disease, a cancer, an ocular disorder, a seizure disorder, a lysosomal storage disease, an amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and CNS inflammation.

In another aspect of the above embodiments, the invention provides any of the aforementioned antibodies for use in transporting one or more compounds across the BBB. In another aspect of the above embodiments, the invention provides use of any of the aforementioned antibodies in the preparation of a medicament for transporting one or more compounds across the BBB.

In one aspect of the above embodiment, a method for transporting a compound across the BBB in a subject is provided, the method comprising exposing any of the aforementioned antibodies to the BBB so that the antibody transports the compound coupled thereto across the BBB. In another such aspect, the BBB is in a human subject. In another such aspect, the dosage and/or frequency of administration is adjusted to reduce the concentration of the antibody to which the red blood cells are exposed. In another such aspect, the method further comprises the step of monitoring the subject for red blood cell depletion. In another such aspect, the antibody coupled to the compound is administered at a therapeutic dose. In one such aspect, the therapeutic dose is TfR-saturated. In another such aspect, administration of the antibody is performed at a calibrated dosage and/or dosage frequency to minimize acute clinical symptoms of antibody administration.

In another aspect of the above embodiment, there is provided a method for increasing the exposure of the CNS of a subject to a compound, the method comprising exposing any of the aforementioned antibodies to the BBB so that the antibody transports the compound coupled thereto across the BBB. In another such aspect, the BBB is in a human subject. In another such aspect, the dosage and/or frequency of administration are adjusted to reduce the concentration of the antibody to which the red blood cells are exposed. In another such aspect, the method further comprises the step of monitoring the red blood cell consumption of the subject. In another such aspect, the antibody coupled to the compound is administered at a therapeutic dose. In one such aspect, the therapeutic dose is TfR-saturated. In another such aspect, the administration of the antibody is carried out at a calibrated dosage and/or dosage frequency to minimize the acute clinical symptoms of antibody administration.

In one aspect of the above embodiment, there is provided a method for increasing the retention of a compound administered to a subject in the CNS, the method comprising exposing any of the aforementioned antibodies to the BBB to increase the retention of the compound in the CNS. In another such aspect, the BBB is in a human subject. In another such aspect, the dosage and/or frequency of administration is adjusted to reduce the concentration of the antibody to which the red blood cells are exposed. In another such aspect, the method further comprises the step of monitoring the subject for red blood cell depletion. In another such aspect, the antibody coupled to the compound is administered at a therapeutic dose. In one such aspect, the therapeutic dose is TfR-saturated. In another such aspect, the administration of the antibody is carried out at a calibrated dosage and/or dosage frequency to minimize the acute clinical symptoms of antibody administration.

In one aspect of the above-mentioned embodiment, there is provided a method for treating a nervous system disease in a mammal, the method comprising treating the mammal with any of the aforementioned antibodies. In one such aspect, the nervous system disease is selected from the group consisting of: neurological conditions, neurodegenerative diseases, cancer, eye conditions, epileptic seizure conditions, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders, and CNS inflammation. In another such aspect, the nervous system disease is in a human subject. In another such aspect, the dosage and/or frequency of administration are adjusted to reduce the concentration of the antibody to which the red blood cells are exposed. In another such aspect, the method further comprises the step of monitoring the red blood cell consumption of the subject. In another such aspect, the antibody coupled to the compound is administered in a therapeutic dose. In one such aspect, the therapeutic dose is TfR-saturated. In another such aspect, the administration of the antibody is carried out with a calibrated dosage and/or dosage frequency to minimize the acute clinical symptoms of antibody administration.

In another embodiment, the invention provides an isolated antibody that binds to human TfR and primate TfR, wherein the antibody does not inhibit the binding of transferrin to TfR, and wherein one or more properties of the antibody are modified to reduce or eliminate the effect of the antibody on reticulocytes and/or reduce the severity or presence of acute clinical symptoms in a subject or mammal treated with the antibody. In one aspect, the binding is specific binding. In another aspect, the antibody also does not inhibit the binding of HFE to TfR. In one aspect, the antibody is a monoclonal antibody. In another aspect, the antibody is a human antibody. In another aspect, the antibody is a humanized antibody. In another aspect, the antibody is a chimeric antibody. In another aspect, the antibody is an antibody fragment that binds to human TfR and primate TfR. In another aspect, the primate TfR is from cynomolgus monkey.

In one aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 31, 33, and 34, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 36, 38, and 39, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 36, 40, and 34, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 42, 43, and 44, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 31, 33, and 34, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 46, 48 and 49, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 52, 54 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 52, 58 and 59, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 62, 63 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 52, 65 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 68, 69 and 70, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 73, 75 and 76, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 79, 81 and 82, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 79, 83 and 84, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 87, 89 and 90, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 93, 95, and 96, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 99, 101, and 102, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L3, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 127, 33, and 34, respectively.

In one aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 37, 38, and 39, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 32, 40, and 34, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 37, 43, and 44, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 47, 48 and 49, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 54 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 58 and 59, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 63 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 65 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 69 and 70, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 74, 75 and 76, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 80, 81 and 82, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 80, 83 and 84, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 88, 89 and 90, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 94, 95 and 96, respectively. In another aspect of the above embodiment, the antibody comprises HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 100, 101 and 102, respectively.

In one aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 29, 30, and 31, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 35, 30, and 36, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 41, 30, and 42, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 29, 30, and 31, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 45, 30, and 46, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 50, 51, and 52, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 56, 57, and 52, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 60, 61, and 62, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 60, 64, and 52, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 66, 67, and 68, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 71, 72, and 73, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 77, 78, and 79, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 85, 86, and 87, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 91, 92, and 93, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 97, 98, and 99, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 29, 30 and 127, respectively.

In one aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 29, 30, and 31, respectively, and HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 35, 30, and 36, respectively, and HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 37, 38, and 39, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 35, 30 and 36, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 32, 40 and 34, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 41, 30 and 42, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 37, 43 and 44, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 29, 30, and 31, respectively, and HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 45, 30, and 46, respectively, and HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 50, 51 and 52, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 54 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 56, 57 and 52, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 58 and 59, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 60, 61 and 62, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 63 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 60, 64 and 52, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 65 and 55, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 66, 67 and 68, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 53, 69 and 70, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 71, 72 and 73, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 74, 75 and 76, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 77, 78 and 79, respectively, and HVR-H1, HVR-H2 and HVR-H comprising the amino acid sequences of SEQ ID NOs: 80, 81 and 82, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 77, 78 and 79, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 80, 83 and 84, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 85, 86 and 87, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 88, 89 and 90, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2 and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 91, 92 and 93, respectively, and HVR-H1, HVR-H2 and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 94, 95 and 96, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 97, 98, and 99, respectively, and HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 100, 101, and 102, respectively. In another aspect of the above embodiment, the antibody comprises HVR-L1, HVR-L2, and HVR-L3 comprising the amino acid sequences of SEQ ID NOs: 29, 30, and 127, respectively, and HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively.

In one aspect of the above embodiments, the antibody comprises at least one HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 or HVR-L3 sequence selected from the group consisting of: an HVR-H1 sequence of SEQ ID NO: 47, 53 or 100; an HVR-H2 sequence of SEQ ID NO: 48, 69 or 101; an HVR-H3 sequence of SEQ ID NO: 49, 76 or 102; an HVR-L1 sequence of SEQ ID NO: 45, 66 or 97; an HVR-L2 sequence of SEQ ID NOs: 30, 67 or 98; and an HVR-L3 sequence of SEQ ID NOs: 46, 68 or 102.

In one aspect of the above embodiments, the antibody comprises (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 7, 8, 9, 10, 15, 16, 17, 18, 20, 25, 26, 27, 28, 108, 114, 120, or 126; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4, 5, 6, 11, 12, 13, 14, 19, 21, 22, 23, 24, 105, 111, 117, or 123; or (c) the VH sequence of (a) and the VL sequence of (b). In one such aspect, the antibody comprises the VL sequence of SEQ ID NO: 4 and the VH sequence of SEQ ID NO: 7. In another such aspect, the antibody comprises the VL sequence of SEQ ID NO: 5 and the VH sequence of SEQ ID NO: 8. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 5 and a VH sequence of SEQ ID NO: 9. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 6 and a VH sequence of SEQ ID NO: 10. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 11 and a VH sequence of SEQ ID NO: 15. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 12 and a VH sequence of SEQ ID NO: 16. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 13 and a VH sequence of SEQ ID NO: 17. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 14 and a VH sequence of SEQ ID NO: 18. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 19 and a VH sequence of SEQ ID NO: 20. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 21 and a VH sequence of SEQ ID NO: 25. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 22 and a VH sequence of SEQ ID NO: 26. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 23 and a VH sequence of SEQ ID NO: 27. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 24 and a VH sequence of SEQ ID NO: 28. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 105 and a VH sequence of SEQ ID NO: 108. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 111 and a VH sequence of SEQ ID NO: 114. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 117 and a VH sequence of SEQ ID NO: 120. In another such aspect, the antibody comprises a VL sequence of SEQ ID NO: 123 and a VH sequence of SEQ ID NO: 126. In another such aspect, the antibody comprises the VH sequence of (a) SEQ ID NO: 108, (b) SEQ ID NO: 114, (c) SEQ ID NO: 120, or (d) SEQ ID NO: 126. In another such aspect, the antibody comprises the VL sequence of (a) SEQ ID NO: 105, (b) SEQ ID NO: 111, (c) SEQ ID NO: 117, or (d) SEQ ID NO: 123.

In one aspect of the above embodiment, the antibody is selected from the group consisting of antibodies 7A4, 8A2, 15D2, 10D11, 7B10, 15G11, 16G5, 13C3, 16G4, 16F6, 7G7, 4C2, 1B12, and 13D4. In one such aspect, the antibody is 7A4. In another such aspect, the antibody is 8A2. In another such aspect, the antibody is 15D2. In another such aspect, the antibody is 10D11. In another such aspect, the antibody is 7B10. In another such aspect, the antibody is 15G11. In another such aspect, the antibody is 16G5. In another such aspect, the antibody is 13C3. In another such aspect, the antibody is 16G4. In another such aspect, the antibody is 16F6. In another such aspect, the antibody is 7G7. In another such aspect, the antibody is 4C2. In another such aspect, the antibody is 1B12. In another such aspect, the antibody is 13D4.

In one aspect of any of the foregoing, the antibody is further affinity matured. In one such aspect, the antibody is selected from the group consisting of: 15G11.v1, 15G11.v2, 15G11.v3, 15G11.v4, 15G11.v5, 7A4.v1; 7A4.v2, 7A4.v3, 7A4.v4, 7A4.v5, 7A4.v6, 7A4.v7, 7A4.v8, 7A4.v9, 7A4.v10, 7A4.v11, 7A4.v12, 7A4.v13, 7A4.v14, 7A4.v15, 7G7.v1, 16F6.v1, 16F6.v2, 16F6.v3, and 16F6.v4. In one such aspect, the antibody is 15G11.v1. In another such aspect, the antibody is 15G11.v2. In another such aspect, the antibody is 15G11.v3. In another such aspect, the antibody is 15G11.v4. In another such aspect, the antibody is 15G11.v5. In another such aspect, the antibody is 7A4.v1. In another such aspect, the antibody is 7A4.v2. In another such aspect, the antibody is 7A4.v3. In another such aspect, the antibody is 7A4.v4. In another such aspect, the antibody is 7A4.v5. In another such aspect, the antibody is 7A4.v6. In another such aspect, the antibody is 7A4.v7. In another such aspect, the antibody is 7A4.v8. In another such aspect, the antibody is 7A4.v9. In another such aspect, the antibody is 7A4.v10. In another such aspect, the antibody is 7A4.v11. In another such aspect, the antibody is 7A4.v12. In another such aspect, the antibody is 7A4.v13. In another such aspect, the antibody is 7A4.v14. In another such aspect, the antibody is 7A4.v15. In another such aspect, the antibody is 7G7.v1. In another such aspect, the antibody is 16F6.v1. In another such aspect, the antibody is 16F6.v2. In another such aspect, the antibody is 16F6.v3. In another such aspect, the antibody is 16F6.v4. In another such aspect, the antibody is 15G11.N52A. In another such aspect, the antibody is 16F6.v4. In another such aspect, the antibody is 15G11.T53A. In another such aspect, the antibody is 16F6.v4. In another such aspect, the antibody is 15G11.W92A. In another such aspect, the antibody is 16F6.v4.

In one aspect of the above embodiment, the antibody is modified at one or more amino acid positions of VH or VL to the amino acids shown in Figures 4E-1 and 4E-2 for the positions. In another aspect of the above embodiment, the antibody comprises a sequence corresponding to the sequence described for any one of the clones in Figures 3 and 4 and Figures 4E-1 and 4E-2 or one or more of these sequences or one or more HVR sequences. In another aspect of the above embodiment, the antibody comprises a VH or VL sequence corresponding to the VH or VL sequence described for any one of the clones in Figures 3 and 4 and Figures 4E-1 and 4E-2. In another aspect of the above embodiment, the antibody comprises one or more HVR sequences corresponding to one or more of those described for any one of the clones in Figures 3 and 4 and Figures 4E-1 and 4E-2.

In one aspect of the above embodiment, the one or more properties of the antibody are selected from the effector functions of the antibody Fc region, the complement activation function of the antibody, and the affinity of the antibody for TfR. In one such aspect, the property is the effector function of the antibody Fc region. In another such aspect, the property is the complement activation function of the antibody. In another such aspect, the property is the affinity of the antibody for TfR. In one such aspect, the effector function or complement activation function is reduced or eliminated relative to a wild-type antibody of the same isotype. In one aspect, the effector function is reduced or eliminated by a method selected from the following: reducing the glycosylation of the antibody, modifying the isotype of the antibody to an isotype that naturally reduces or eliminates the effector function, and modifying the Fc region.

In one such aspect, effector function is reduced or eliminated by reducing glycosylation of the antibody. In one such aspect, glycosylation of the antibody is reduced by a method selected from the group consisting of: producing the antibody in an environment that does not permit wild-type glycosylation; removing carbohydrate groups already present in the antibody; and modifying the antibody such that wild-type glycosylation does not occur. In one such aspect, glycosylation of the antibody is reduced by producing the antibody in an environment that does not permit wild-type glycosylation, such as in an environment where the antibody is produced or synthetically prepared in a non-mammalian cell production system. In one such aspect, the antibody is produced in a non-mammalian cell production system. In another such aspect, the antibody is produced synthetically. In another such aspect, glycosylation of the antibody is reduced by modifying the antibody such that wild-type glycosylation does not occur, such as where the Fc region of the antibody comprises a mutation at position 297 such that the wild-type asparagine residue at that position is replaced with another amino acid that interferes with glycosylation at that position.

In another such aspect, effector function is reduced or eliminated by at least one modification of the Fc region. In one such aspect, effector function or complement activation function is reduced or eliminated by deleting all or part of the Fc region or by engineering the antibody so that it does not comprise an Fc region or non-Fc region that is competent for effector function or complement activation function. In another such aspect, at least one modification of the Fc region is selected from: an Fc region point mutation that weakens 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, 3 24, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438, and 439; an Fc region point mutation that impairs binding to C1q at a position selected from the group consisting of: 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. In one such aspect, the mutation is a point mutation in the Fc region that impairs binding to C1q at a position selected from the group consisting of: 270, 322, 329, and 321. In another such aspect, the modification is elimination of some or all of the Fc region. In another such aspect, complement priming function is reduced or eliminated by deleting all or part of the Fc region or by engineering the antibody so that it does not contain an Fc region involved in the complement pathway. In one such aspect, the antibody is selected from a Fab or a single-chain antibody. In another such aspect, the non-Fc region of the antibody is modified to reduce or eliminate activation of the complement pathway by the antibody. In one such aspect, the modification is a point mutation in the CH1 region that weakens binding to C3. In one such aspect, the point mutation is at position 132 (e.g., see Vidarte et al., (2001) J. Biol. Chem. 276(41):38217-38223).

In one aspect, the half-life of an antibody is increased by modifications in the FcRn binding region. In one aspect, the modification is an amino acid replacement selected from the group consisting of: 251, 256, 285, 290, 308, 314, 385, 389, 428, 434, 436, 238, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434. In one aspect, the modification is a replacement selected from the group consisting of: M252Y, S254T, T256E, N434A, and Y436I.

In one aspect, the antibody is combined with another compound that reduces or helps reduce the impact on reticulocyte levels or acute clinical symptoms. In one such aspect, the another compound protects reticulocytes from antibody-related depletion or supports the growth, development, or rebuilding of reticulocytes. In another such aspect, the another compound is selected from erythropoietin (EPO), iron supplements, vitamin C, folic acid, and vitamin B12, or is red blood cells or reticulocytes.

In one aspect of the above embodiment, the antibody has reduced affinity for TfR as measured relative to a wild-type antibody of the same isotype that does not have reduced affinity for TfR. In one such aspect, the antibody has a KD or IC50 for TfR of about 1 pM to about 100 μM. In another aspect, the dosage amount and/or frequency of administration of the antibody is adjusted to reduce the concentration of antibody to which red blood cells are exposed.

In one aspect of the above embodiment, the antibody is coupled to a therapeutic compound. In another aspect of the above embodiment, the antibody is coupled to an imaging agent or a label. In one such aspect, the antibody is a multispecific antibody, and the therapeutic compound optionally forms a part of the multispecific antibody. In one such aspect, the multispecific antibody comprises a first antigen binding site that binds to TfR and a second antigen binding site that binds to a brain antigen. In one such aspect, the brain antigen is selected from the group consisting of: beta-secretase 1 (BACE1), Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E (ApoE), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR) and caspase 6. In another such aspect, the multispecific antibody binds both TfR and BACE1. In another such aspect, the multispecific antibody binds both TfR and Aβ. In another such aspect, the therapeutic compound is a neurological disease drug.

In one aspect of the above embodiments, the present invention provides isolated nucleic acids encoding any of the aforementioned antibodies. In another aspect, the present invention provides host cells comprising the nucleic acids. In another aspect, the present invention provides methods for preparing any of the aforementioned antibodies, the methods comprising culturing the host cells to produce the antibodies, and optionally further comprising recovering the antibodies from the host cells.

In one aspect of the above embodiments, the invention provides a pharmaceutical formulation comprising any of the aforementioned antibodies and a pharmaceutically acceptable carrier.

In one aspect of the above embodiments, the present invention provides any of the aforementioned antibodies for use as a medicament. In another aspect of the above embodiments, the present invention provides use of any of the aforementioned antibodies in the preparation of a medicament for treating a neurological disease. In one such aspect, the neurological disease is selected from the group consisting of a neurological disorder, a neurodegenerative disease, a cancer, an ophthalmic disorder, a seizure disorder, a lysosomal storage disease, an amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and CNS inflammation.

In another aspect of the above embodiments, the invention provides any of the aforementioned antibodies for use in treating a neurological disease. In one such aspect, the neurological disease is selected from the group consisting of a neurological disorder, a neurodegenerative disease, a cancer, an ocular disorder, a seizure disorder, a lysosomal storage disease, an amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and CNS inflammation.

In another aspect of the above embodiments, the invention provides any of the aforementioned antibodies for use in transporting one or more compounds across the BBB. In another aspect of the above embodiments, the invention provides use of any of the aforementioned antibodies in the preparation of a medicament for transporting one or more compounds across the BBB.

In one aspect of the above embodiment, a method for transporting a compound across the BBB in a subject is provided, the method comprising exposing any of the aforementioned antibodies to the BBB so that the antibody transports the compound coupled thereto across the BBB. In another such aspect, the BBB is in a human subject. In another such aspect, the dosage and/or frequency of administration is adjusted to reduce the concentration of the antibody to which the red blood cells are exposed. In another such aspect, the method further comprises the step of monitoring the subject for red blood cell depletion. In another such aspect, the antibody coupled to the compound is administered at a therapeutic dose. In one such aspect, the therapeutic dose is TfR-saturated. In another such aspect, administration of the antibody is performed at a calibrated dosage and/or dosage frequency to minimize acute clinical symptoms of antibody administration.

In another aspect of the above embodiment, there is provided a method for increasing the exposure of the CNS of a subject to a compound, the method comprising exposing any of the aforementioned antibodies to the BBB so that the antibody transports the compound coupled thereto across the BBB. In another such aspect, the BBB is in a human subject. In another such aspect, the dosage and/or frequency of administration are adjusted to reduce the concentration of the antibody to which the red blood cells are exposed. In another such aspect, the method further comprises the step of monitoring the red blood cell consumption of the subject. In another such aspect, the antibody coupled to the compound is administered at a therapeutic dose. In one such aspect, the therapeutic dose is TfR-saturated. In another such aspect, the administration of the antibody is carried out at a calibrated dosage and/or dosage frequency to minimize the acute clinical symptoms of antibody administration.

In one aspect of the above embodiment, there is provided a method for increasing the retention of a compound administered to a subject in the CNS, the method comprising exposing any of the aforementioned antibodies to the BBB to increase the retention of the compound in the CNS. In another such aspect, the BBB is in a human subject. In another such aspect, the dosage and/or frequency of administration is adjusted to reduce the concentration of the antibody to which the red blood cells are exposed. In another such aspect, the method further comprises the step of monitoring the subject for red blood cell depletion. In another such aspect, the antibody coupled to the compound is administered at a therapeutic dose. In one such aspect, the therapeutic dose is TfR-saturated. In another such aspect, the administration of the antibody is carried out at a calibrated dosage and/or dosage frequency to minimize the acute clinical symptoms of antibody administration.

In one aspect of the above-mentioned embodiment, there is provided a method for treating a nervous system disease in a mammal, the method comprising treating the mammal with any of the aforementioned antibodies. In one such aspect, the nervous system disease is selected from the group consisting of: neurological conditions, neurodegenerative diseases, cancer, eye conditions, epileptic seizure conditions, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders and CNS inflammation. In another such aspect, the nervous system disease is in a human subject. In another such aspect, the dosage and/or frequency of administration are adjusted to reduce the concentration of the antibody to which the red blood cells are exposed. In another such aspect, the method further comprises the step of monitoring the red blood cell consumption of the subject. In another such aspect, the antibody coupled to the compound is administered in a therapeutic dose. In one such aspect, the therapeutic dose is TfR-saturated. In another such aspect, the administration of the antibody is carried out with a calibrated dosage and/or dosage frequency to minimize the acute clinical symptoms of antibody administration.

In another embodiment, the invention provides an isolated antibody that binds to the same epitope on TfR as an antibody selected from the group consisting of antibodies 7A4, 8A2, 15D2, 10D11, 7B10, 15G11, 16G5, 13C3, 16G4, 16F6, 7G7, 4C2, 1B12, 13D4, 15G11.v1, 15G11.v2, 15G11.v3, 15G11.v 4, 15G11.v5, 7A4.v1; 7A4.v2, 7A4.v3, 7A4.v4, 7A4.v5, 7A4.v6, 7A4.v7, 7A4.v8, 7A4.v9, 7A4.v1 0, 7A4.v11, 7A4.v12, 7A4.v13, 7A4.v14, 7A4.v15, 7G7.v1, 16F6.v1, 16F6.v2, 16F6.v3, 16F6.v4 15G11.N52A, 15G11.T53A and 15G11W92A.

In another embodiment, a method is provided for reducing the clearance of a compound administered to a subject, wherein the compound is conjugated to an antibody with low affinity for TfR to reduce the clearance of the compound, and wherein the reduction in red blood cell levels in the subject is reduced or eliminated when the compound-conjugated antibody is administered to the subject.

In another embodiment, a method is provided for optimizing the pharmacokinetics and/or pharmacodynamics of a compound effective in the CNS of a subject, wherein the compound is conjugated to an antibody that binds TfR with low affinity, and the antibody is selected such that its affinity for TfR upon conjugation to the compound results in an amount of the antibody conjugated to the compound being transported across the BBB, which optimizes the pharmacokinetics and/or pharmacodynamics of the compound in the CNS, wherein a decrease in red blood cell levels in the subject upon administration of the compound-conjugated antibody to the subject is reduced or eliminated.

It will be understood that any of the foregoing methods and compositions of the invention may be combined with each other and/or with other aspects of the invention described in the specification herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the three-dimensional crystal structure of the TfR dimer in complex with Tf based on pdb file 3SM9. The non-Tf-binding apical region of TfR is labeled.

Figures 2A-2B show FACS analysis of mouse hybridoma parental clone supernatants binding to human and cynomolgus monkey TfR transiently expressed in 293 cells in the presence of 1 μM human holo-Tf. Unless otherwise indicated, the filled gray trace in each figure is background from the detection antibody, the medium gray trace is binding to 293 cells endogenously expressing basal levels of human TfR, the thick black trace represents binding to transiently expressed human TfR, and the thin gray trace represents binding to transiently expressed cynomolgus monkey TfR.

Figure 2C shows the results of a human/cynomolgus monkey cross-reactive antibody competition assay as described in Example 1. Nine out of fourteen clones were found to block binding of the tip-binding antibody displayed on phage.

Figures 3A-1, 3A-2, 3B-1, 3B-2, 3C-1, 3C-2, 3D-1 and 3D-2 show the heavy and light chain variable region sequences of hybridoma clones that bind to the apical and non-apical regions of TfR. The sequences can be further subdivided into classes I-III (apical binders) and class IV (non-apical binders) based on epitope and sequence similarity. HVRs according to Kabat are underlined.

Figures 4A-1, 4A-2, 4B-1, 4B-2, 4C-1, 4C-2, 4D-1 and 4D-2 show the alignment of the humanized sequences for (A) 15G11, (B) 7A4/8A2, (C) 7G7 and (D) 16F6. Each mouse light or heavy variable domain sequence (second row) is aligned with the closest human germline or consensus variable domain (first row). The humanized version of each antibody is shown below (third row). The differences from the human germline or consensus sequence are shaded. The HVR sequences transplanted onto the human framework are boxed. The CDR definitions according to Kabat are shown.

Figures 4E-1 and 4E-2 show that with respect to class I-III groups of antibodies, variant forms of antibodies with modifications at one or more residues in the FRs retain affinity and binding specificity.

Figure 5 shows the binding of hu7A4.v15, hu15G11.v5 and hu7G7.v1 to huTfR in the presence of 6.3 μM holo-Tf. Antibody binding to immobilized huTfR is shown in the presence (open symbols and dashed line) or absence (filled symbols and solid line) of 6.3 μM holo-Tf.

Figures 6A-B show the results of the HFE-HuTfR binding and HFE blocking assays described in Example 1. Figure 6A shows antibody binding to increasing concentrations of huTfR captured by immobilized HFE. Figure 6B shows huTfR binding to immobilized HFE in the presence of increasing concentrations of antibody.

Figures 7A-B show binding assays of 15G11.v5 and 7A4.v5 IgG and Fab Ala variants to cynomolgus monkey and human TfR, demonstrating the effect of Ala mutations in CDR-L3 and CDR-H3 of each antibody on affinity to immobilized human or cynomolgus monkey TfR, as determined by ELISA binding as IgG and by SPR analysis as IgG or Fab, as described in Example 2.

8A-B and 9A-B show the results of experiments to determine the influence of effector function status on ADCC activity of anti-human TfR ("anti-hTFR") antibodies in primary human bone marrow mononuclear cells or in a human erythroblast line, as described in Example 4.

FIG10 shows the dosing and sampling scheme for the primate study described in Example 5.

Figures 11A-11B show the pharmacokinetic results of the experiments described in Example 5, specifically, individual or group mean anti ^-TfR1 /BACE1, anti-TfR2/BACE1, and anti-gD serum concentrations in serum (Figure 11A) and CSF (Figure 11B) over time following a single IV bolus administration of ³⁰ mg/kg in cynomolgus monkeys.

Figures 12A-12E show the pharmacodynamic results of the experiments described in Example 5, specifically, individual and group mean anti-TfR ¹ / BACE1, anti-TfR ² / BACE1, and anti-gD plasma (A) or CSF (BE) concentrations over time in cynomolgus monkeys after a single IV bolus of 30 mg/kg. The upper graph shows Aβ 1-40 levels in plasma (Figure 12A) and CSF (Figure 12B), while the lower graph shows soluble APPα levels (Figure 12C), soluble APPβ levels (Figure 12D), and sAPPβ/sAPPα ratio (Figure 12E) over time.

Figures 13A-13D show the results of hematological sampling performed during the study described in Example 5. At each indicated time point, total reticulocytes (Figure 13A), erythrocytes (Figure 13B), hemoglobin (Figure 13D), and the percentage of immature reticulocytes in the total reticulocyte pool (Figure 13C) were measured using standard techniques.

FIG14 shows the dosing and sampling scheme for the primate study described in Example 6.

Figures 15A-15B show the pharmacodynamic results (A) and brain antibody concentrations (B) of the experiments described in Example 6. Specifically, Figure 15A shows individual and group average anti-TfR ^{1 / BACE1, anti-TfR 2 / BACE1, anti-gD, and sAPPβ/sAPPα anti-BACE1 quantification in CSF over time after a single IV bolus of 30 mg/kg in cynomolgus monkeys. Figure 15B shows individual anti-TfR 1 / BACE1, anti-TfR 2 / BACE1} , anti-gD, and anti-BACE1 concentrations of antibodies in different brain regions 24 hours after dosing.

Figures 16A-B show the light and heavy chain amino acid sequences of the anti-BACE1 clone YW412.8 obtained from a natural diversity phage display library species used for the first experiment, as well as the affinity matured form of YW412.8. Figure 16A shows the variable light chain (VL) sequence alignment (SEQ ID NOs. 132-137). Figure 16B shows the variable heavy chain (VH) sequence alignment (SEQ ID Nos. 138-139). In both figures, the HVR sequence of each clone is represented by a boxed area, the first box representing HVR-L1 (Figure 16A) or HVR-H1 (Figure 16B), the second box representing HVR-L2 (Figure 16A) or HVR-H2 (Figure 16B), and the third box representing HVR-L3 (Figure 16A) or HVR-H3 (Figure 16B).

Figures 17A-B show the light and heavy chain amino acid sequences of anti-BACE1 antibody clone Fab 12 obtained from a naive synthetic diversity phage display library, as well as affinity-matured forms of Fab 12. Figure 17A shows a light chain sequence alignment (SEQ ID NOs. 140-143). Figure 17B shows a heavy chain sequence alignment (SEQ ID NO. 144). In both figures, the HVR sequences of each clone are represented by boxed regions, with the first box representing HVR-L1 (Figure 17A) or HVR-H1 (Figure 17B), the second box representing HVR-L2 (Figure 17A) or HVR-H2 (Figure 17B), and the third box representing HVR-L3 (Figure 17A) or HVR-H3 (Figure 17B).

Figures 18A-B show the heavy chain (Figure 18A; SEQ ID NO. 145) and light chain (Figure 18B; SEQ ID NO. 146) of an exemplary anti-Aβ antibody.

FIG19 shows the pharmacokinetic properties of anti- ^TfR1 /BACE1, anti- ^Tf52A /BACE1, and anti-TfR53A/BACE1 described in Example 5. FIG19 shows the pharmacokinetic properties of anti-TfR1/BACE1, anti-TfR52A/BACE1, and anti- ^TfR53A /BACE1 described in Example 5.

FIG20 shows the pharmacokinetic properties of the murine IgG2a anti- ^TfRD /BACE1 and anti-gD antibodies with the Fc effector function LALAPG mutation described in Example 7.

21 shows total and immature reticulocyte counts 24 hours after administration of a 50 mg/kg dose of murine IgG2a anti- ^TfRD /BACE1 and anti-gD antibodies with Fc effector function LALAPG mutation as described in Example 7.

Figure 22 shows the total reticulocyte counts in mice 24 hours after administration of a 50 mg/kg dose of anti-TfR ^52A /BACE1(N297G), anti-TfR ^52A /BACE1(LALAPG), anti-TfR ⁵² A/BACE1(LALAPG/YTE), TfR ⁵² A/BACE1(LALAPG/AI) antibodies in mice with human transferrin receptor knock-in as described in Example 8.

Figure 23 shows the results of experiments to evaluate the effect of effector function status on the ADCC activity of anti-TfR/gD, anti-TfR/BACE1(N297G), anti-TfR/BACE1(LALAPG), anti-TfR/BACE1(N297G/434A/436I) and anti-TfR/BACE1(LALAPG/YTE) antibodies in primary human bone marrow mononuclear cells or in human erythroblast lines, as described in Example 8.

Figure 24 shows the heavy and light chain variable region sequences of 1511Gv.5 (light chain - SEQ ID NO: 105 and heavy chain - SEQ ID NO: 108) and the heavy and light chain variable region sequences of the following affinity variants: 15G11.52A (light chain - SEQ ID NO: 105 and heavy chain - SEQ ID NO: 153), 15G11.53A (light chain - SEQ ID NO: 105 and heavy chain - SEQ ID NO: 154) and 15G11.92A (light chain - SEQ ID NO: 151 and heavy chain - SEQ ID NO: 108). HVRs according to Kabat are underlined.

FIG25 shows a competition assay between 15G11v.5 and anti-TfR ^C12 as described in Example 1.

Detailed description of the embodiment of the invention

1. Definition

"Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can often be expressed in terms of a dissociation constant (KD, which is the ratio of the rate of dissociation of X from Y (kd or Koff) to the rate of association of X with Y (ka or kon)). An alternative measure of the affinity of one or more antibodies for their target is their half-maximal inhibitory concentration (IC50), a measure of how much antibody is required to inhibit 50% of the binding of a known ligand to the antibody target. Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described herein. "Blood-brain barrier" or "BBB" refers to the physiological barrier between the peripheral circulation and the brain and spinal cord (i.e., CNS), which is formed by tight junctions in the cortical membranes within the brain capillaries, creating a tight barrier that limits the transport of molecules (even very small molecules such as urea (60 daltons)) into the brain. The blood-brain barrier in the brain, the blood-spinal barrier in the spinal cord, and the blood-retinal barrier in the retina are capillary barriers connected in the CNS and are collectively referred to herein as blood-brain barrier or BBB. The BBB also includes a blood-CSF barrier (choroid plexus), wherein the barrier is composed of ependymal cells rather than capillary endothelial cells.

The terms "β-amyloid,""β-amyloid,""Abeta,""amyloidβ," and "Aβ" are used interchangeably herein and refer to fragments of the amyloid precursor protein ("APP") produced when β-secretase 1 ("BACE1") cleaves APP, as well as modifications, fragments, and any functional equivalents thereof, including, but not limited to, Aβ _1-40 and Aβ _1-42 . Aβ is known to exist in monomeric form and to associate to form oligomers and fibrillar structures, which can be found as constituent members of amyloid plaques. The structure and sequence of Aβ peptides are well known to those of ordinary skill in the art, and methods for producing such peptides or extracting such peptides from brain and other tissues are described, for example, in Glenner and Wong, Biochem Biophys Res. Comm. 129: 885-890 (1984). In addition, Aβ peptides are also commercially available in various forms.

"Anti-Aβ immunoglobulin," "anti-Aβ antibody," and "antibody that binds to Aβ" are used interchangeably herein and refer to an antibody that specifically binds to human Aβ. A non-limiting example of an anti-Aβ antibody is crenezumab. Other non-limiting examples of anti-Aβ antibodies are solanezumab, bapineuzumab, gantenerumab, aducanumab, ponezumab, and any of the anti-Aβ antibodies disclosed in the following publications: WO2000162801, WO2002046237, WO2002003911, WO2003016466, WO2003016467, WO200 03077858, WO2004029629, WO2004032868, WO2004032868, WO2004108895, WO2005028511, WO200603 9470, WO2006036291, WO2006066089, WO2006066171, WO2006066049, WO2006095041, WO2009027105.

The terms "crenezumab" and "MABT5102A" are used interchangeably herein and refer to a specific anti-Aβ antibody that binds monomeric, oligomeric, and fibrillar forms of Aβ and is associated with CAS Registry Number 1095207. In one embodiment, the antibody comprises the sequence shown in Figures 18A and 18B.

"ApoE4 carrier" or "ApoE4 carrier" is used interchangeably with "apoE4 positive" or "ApoE4 positive" to refer to an individual who has at least one apoE4 (or "ApoE4") allele. Individuals who have zero ApoE4 alleles are referred to herein as "ApoE4 negative" or "non-ApoE4 carriers." See also Prekumar, et al., 1996, Am. J Pathol. 148: 2083-95.

The term "cerebral vasogenic edema" refers to an excessive accumulation of intravascular fluid or protein within brain cells or in the extracellular space. For example, cerebral vasogenic edema can be detected by brain MRI, including but not limited to FLAIR MRI, and can be asymptomatic ("asymptomatic vasogenic edema") or associated with neurological disorders such as confusion, dizziness, vomiting, and lethargy ("symptomatic vasogenic edema") (see Sperling et al., Alzheimer's & Dementia, 7:367, 2011).

The term "cerebral hemorrhage" refers to intracranial hemorrhage or bleeding in the brain in an area greater than about 1 cm in diameter. Cerebral hemorrhage can be detected by, for example, brain MRI (which includes but is not limited to T2*-weighted GRE MRI) and can be asymptomatic ("asymptomatic hemorrhage") or associated with symptoms such as temporary or permanent local motor or sensory impairment, ataxia, aphasia, and dysarthria ("symptomatic hemorrhage") (e.g., see Chalela JA, Gomes J. Expert Rev. Neurother. 2004 4: 267, 2004 and Sperling et al. Alzheimer's & Dementia, 7: 367, 2011).

The term "cerebral microhemorrhage" refers to intracranial hemorrhage or bleeding in the brain that is less than about 1 cm in diameter. Cerebral microhemorrhages can be detected, for example, by brain MRI (which includes but is not limited to T2*-weighted GREMRI) and can be asymptomatic ("asymptomatic microhemorrhages") or associated with symptoms such as temporary or permanent focal motor or sensory impairment, ataxia, aphasia, and dysarthria ("symptomatic microhemorrhages"). For example, see Greenberg, et al., 2009, Lancet Neurol. 8: 165-74.

The term "sulcal effusion" refers to an overflow of fluid in the sulci or troughs of the brain. Sulcal effusion can be detected, for example, by brain MRI, including but not limited to FLAIR MRI. See Sperling et al., Alzheimer's & Dementia, 7:367, 2011.

The term "superficial siderosis of the central nervous system" refers to bleeding or hemorrhage into the subarachnoid space of the brain and can be detected, for example, by brain MRI, including but not limited to T2*-weighted GRE MRI. Symptoms indicative of superficial siderosis of the central nervous system include sensorineural deafness, cerebellar ataxia, and pyramidal signs. See Kumara-N, Am J Neuroradiol. 31:5, 2010.

The term "amyloidosis" as used herein refers to a group of diseases and conditions caused by or associated with amyloid or amyloid-like proteins, and includes, but is not limited to, diseases or conditions caused by the presence or activity of amyloid-like proteins in a monomeric, fibrillar, or multimeric state, or any combination of the three, including amyloid plaques. Such diseases include, but are not limited to, secondary amyloidosis and age-related amyloidosis, such as diseases including, but not limited to, neurological diseases such as Alzheimer's disease ("AD"), diseases or conditions characterized by congenital memory loss, such as, for example, mild cognitive impairment (MCI), Lewy body dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam Parkinson-Demential complex and other diseases based on or associated with amyloid-like proteins, such as progressive supranuclear palsy, multiple sclerosis, Creutzfeld Jacob disease, Parkinson's disease, HIV-related dementia, ALS (amyotrophic lateral sclerosis), inclusion-body myositis, and other diseases. Myositis (IBM), adult onset diabetes, endocrine tumors and senile cardiac amyloidosis, as well as a variety of eye diseases, including macular degeneration, drusen-related optic neuropathy, glaucoma, and cataracts due to β-amyloid deposition.

Glaucoma is a group of optic nerve diseases that involve the loss of retinal ganglion cells (RGCs) with a characteristic pattern of optic neuropathy. RGCs are nerve cells that transmit visual signals from the eye to the brain. Two major enzymes in the apoptosis process, namely, caspase-3 and caspase-8, are activated in the process of causing RGCs apoptosis. Caspase-3 cleaves amyloid precursor protein (APP) to produce neurotoxic fragments, including Aβ. Without the protective effect of APP, the accumulation of Aβ in the retinal ganglion cell layer leads to the death of RGCs and irreversible vision loss.

Glaucoma is usually, but not always with increased intraocular pressure, and it may be the result of blocking aqueous humor circulation or its discharge. Although the intraocular pressure that rises is the main risk factor for developing glaucoma, the intraocular pressure threshold that plays a decisive role for causing glaucoma can not be defined. This damage may also be caused by the health problem of the poor blood supply to the important optic nerve fibers, the weakness in the neural structure and/or the nerve fibers themselves. Untreated glaucoma causes permanent optic nerve damage and the visual field loss that causes, and this may progress to blindness.

The term "mild Alzheimer's disease" or "mild AD" as used herein (eg, "a patient diagnosed with mild AD") refers to a stage of AD characterized by an MMSE score of 20-26.

The term "mild to moderate Alzheimer's disease" or "mild to moderate AD" as used herein includes both mild and moderate AD and is characterized by an MMSE score of 18-26.

The term "moderate Alzheimer's disease" or "moderate AD" as used herein (eg, "a patient diagnosed with moderate AD") refers to a stage of AD characterized by an MMSE score of 18-19.

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

"Blood-brain barrier receptors" (abbreviated herein as "BBB-R") are transmembrane receptor proteins expressed on brain endothelial cells that are able to transport molecules across the blood-brain barrier. Examples of BBB-Rs include, but are not limited to, transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF-R), low-density lipoprotein receptor, including but not limited to low-density lipoprotein receptor-related protein 1 (LRP1) and low-density lipoprotein receptor-related protein 8 (LRP8), glucose transporter 1 (Glut1), and heparin-binding epidermal growth factor-like growth factor (HB-EGF). An exemplary BBB-R herein is transferrin receptor (TfR).

The term "transferrin receptor" or "TfR" is used herein to refer to any native TfR from any vertebrate source, unless otherwise indicated, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats). The term includes "full-length," unprocessed TfR as well as any form of TfR that results from processing in the cell. The term also includes naturally occurring TfR variants, e.g., splice variants or allelic variants. TfR is a transmembrane glycoprotein (molecular weight approximately 180,000) composed of two disulfide-bonded subunits (each with an apparent molecular weight of approximately 90,000) that are involved in iron uptake in vertebrates. In one embodiment, the TfR herein is human TfR ("hTfR") comprising the amino acid sequence set forth, for example, in Schneider et al., Nature 311:675-678 (1984) (SEQ ID NO: 1). In another embodiment, the TfR herein is a primate TfR ("pTfR") comprising the amino acid sequence given in Genbank reference AFD18260.1 (SEQ ID NO: 2). For comparison, the mouse TfR sequence can be found in Genbank reference AAH54522.1 (SEQ ID NO: 3).

"Nervous system disease" as used herein refers to a disease or condition that affects the CNS and/or has a cause in the CNS. Exemplary CNS diseases or conditions include, but are not limited to, neuropathy, amyloidosis, cancer, eye diseases or conditions, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, seizures, behavioral disorders, and lysosomal storage diseases. For the purposes of this application, it is understood that the CNS includes the eye, which is typically isolated from the rest of the body by the blood-retinal barrier. Specific examples of nervous system diseases include, but are not limited to, neurodegenerative diseases (including, but not limited to, Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, tauopathies (including, but not limited to, Alzheimer's disease and supranuclear palsy), prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome). syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease and fatal familial insomnia), bulbar palsy, motor neuron disease, and nervous system heterodegenerative disorders (including but not limited to, Canavan disease, Huntington's disease, neuronal ceroid-lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementias (including, but not limited to, Pick's disease and spinocerebellar ataxia), cancers (e.g., cancers of the CNS, including brain metastases from cancers elsewhere in the body).

"Nervous system disease drugs" are drugs or therapeutic agents for treating one or more nervous system diseases. Nervous system disease drugs of the present invention include, but are not limited to, antibodies, peptides, proteins, natural ligands of one or more CNS targets, modified forms of natural ligands of one or more CNS targets, aptamers, inhibitory nucleic acids (i.e., small inhibitory RNA (siRNA) and short hairpin RNA (shRNA)), ribozymes, and small molecules, or active fragments of any of the above. Exemplary nervous system disease drugs of the present invention are described herein and include, but are not limited to, antibodies, aptamers, proteins, peptides, inhibitory nucleic acids and small molecules and active fragments of any of the above, which are themselves or specifically recognize and/or act on (i.e., inhibit, activate, or detect) CNS antigens or target molecules, such as, but not limited to, amyloid precursor protein or a portion thereof, amyloid beta, beta-secretase, gamma-secretase, tau, alpha-synuclein, parkin, huntingtin, DR6, presenilin, ApoE, glioma or other CNS cancer markers, and neurotrophins. Non-limiting examples of neurological disease drugs and the diseases that they can be used to treat are provided in Table 1 below:

Table 1: Non-limiting examples of neurological disease drugs and corresponding diseases that can be treated with them

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 to which a labeling moiety is attached that allows detection.

"CNS antigens" or "brain antigens" are antigens expressed in the CNS, including the brain, that can be targeted by antibodies or small molecules. Examples of such antigens include, but are not limited to, beta-secretase 1 (BACE1), amyloid beta (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), interleukin 6 receptor (IL6R), TNF receptor 1 (TNFR1), interleukin 1 beta (IL1β) and caspase 6. In one embodiment, the antigen is BACE1.

Unless otherwise indicated, the term "BACE1" as used herein refers to any native β-secretase 1 (also known as β-amyloid precursor protein cleaving enzyme 1, membrane-associated aspartic protease 2, memapsin 2, aspartyl protease 2, or Asp2) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term includes "full-length," unprocessed BACE1 as well as any form of BACE1 produced by intracellular processing. The term also includes naturally occurring variants of BACE1, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary BACE1 polypeptide includes the sequence of human BACE1 isoform A, as reported in Vassar et al., Science 286:735-741 (1999), which is incorporated herein by reference in its entirety. There are several other isoforms of human BACE1, including isoforms B, C, and D. See UniProtKB/Swiss-Prot entry P56817, which is incorporated herein by reference in its entirety.

The terms "anti-β-secretase antibody,""anti-BACE1antibody,""antibody that binds to β-secretase," and "antibody that binds to BACE1" refer to antibodies that bind to BACE1 with sufficient affinity so that the antibody can be used as a diagnostic and/or therapeutic agent in targeting BACE1. In one embodiment, the extent of binding of the anti-BACE1 antibody to unrelated, non-BACE1 proteins is less than about 10% of the binding of the antibody to BACE1 (as measured by, for example, radioimmunoassay (RIA)). In certain embodiments, the dissociation constant (Kd) of the antibody that binds to BACE1 is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 ⁻⁸ M or less, e.g., 10 ⁻⁸ M to 10 ⁻¹³ M, e.g., 10 ⁻⁹ M to 10 ⁻¹³ M). In certain embodiments, the anti-BACE1 antibody binds to an epitope of BACE1 that is conserved among BACE1 from different species and isotypes. In one embodiment, an antibody is provided that binds to an epitope on BACE1 that is bound by the anti-BACE1 antibody YW412.8.31. In other embodiments, an antibody is provided that binds to an exosite within BACE1 that is located in the catalytic domain of BACE1. In one embodiment, antibodies are provided that compete for binding to BACE1 with the peptides identified in Kornacker et al., Biochem. 44: 11567-11573 (2005), which is incorporated herein by reference in its entirety (i.e., peptides 1, 2, 3, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, 4, 5, 6, 5-10, 5-9, scrambled, Y5A, P6A, Y7A, F8A, I9A, P10A, and L11A). Exemplary BACE1 antibody sequences are shown in Figures 15A-B and 16A-B. One exemplary antibody herein comprises the variable domains of antibody YW412.8.31 (eg, as in Figures 15A-B).

A "native sequence" protein herein refers to a protein comprising the amino acid sequence of a protein found in nature, including naturally occurring protein variants. The term, when used herein, includes proteins isolated from their natural sources or recombinantly prepared proteins.

The term "antibody" is used herein in the broadest sense and includes a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity.

"Antibody fragment" refers to a molecule that comprises, in addition to an intact antibody, a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments are well known in the art (see, e.g., Nelson, MAbs (2010) 2(1):77-83), and include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , and Fv; diabodies; linear antibodies; single-chain antibody molecules, including but not limited to single-chain variable fragments (scFv), fusions of light and/or heavy chain antigen-binding domains with or without a linker (and optionally in tandem); and monospecific or multispecific antigen-binding molecules formed from antibody fragments (including but not limited to multispecific antibodies constructed from multiple variable domains lacking an Fc region).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., except for possible variants (e.g., comprising naturally occurring mutations, or the variants may occur during the production of monoclonal antibodies, such variants typically being present in smaller amounts), the individual antibodies comprising the group are identical and/or bind to the same epitope. Compared to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the characteristic that the antibody is obtained from a substantially homogeneous antibody group and should not be interpreted as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present invention can be prepared by a variety of techniques, including, but not limited to, the hybridoma method (e.g., see Kohler et al., Nature, 256: 495 (1975)), recombinant DNA methods (e.g., see U.S. Patent No. 4,816,567), phage display methods (e.g., using the techniques described in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991)), and methods using transgenic animals containing all or part of the human immunoglobulin loci, which methods and other exemplary methods for preparing monoclonal antibodies are described herein. Specific examples of monoclonal antibodies herein include chimeric antibodies, humanized antibodies, and human antibodies, including antigen-binding fragments thereof.

Monoclonal antibodies herein specifically include: "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass; and fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Nat. Acad. Sci. USA, 81: 6851-6855 (1984)).

For the purposes of this article, an "acceptor human framework" is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may comprise changes in the amino acid sequence. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the sequence of the VL acceptor human framework is identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

"Human consensus framework" refers to a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of a human immunoglobulin VL or VH sequence is from a subtype of variable domain sequences. Generally, the subtype of the sequence is a subtype as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, the subtype is subtype κI as described in Kabat et al. (see above). In one embodiment, for VH, the subtype is subtype III as described in Kabat et al. (see above).

The "humanized" form of non-human (e.g., mouse) antibodies refers to a chimeric antibody comprising a minimal sequence derived from a non-human antibody. For the main part, a humanized antibody is a human antibody (acceptor antibody), wherein the residues in the acceptor hypervariable region are replaced by hypervariable region residues of a non-human species (donor antibody) (such as mouse, rat, rabbit, or non-human primate) with desired specificity, affinity, and ability. For example, in certain embodiments, a humanized antibody will comprise substantially all of at least one, typically two variable domains, wherein all or substantially all of HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of framework regions (FRs) correspond to those of a human antibody. In some cases, the FR residues of human immunoglobulin (HIG) are replaced by corresponding non-human residues. In addition, humanized antibodies may be included in residues that do not exist in the acceptor antibody or in the donor antibody. These modifications are carried out to further improve antibody performance. In certain embodiments, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human antibody, and all or substantially all of the FRs are those of a human antibody, except that the FRs are replaced as described above. A humanized antibody optionally will also comprise at least a portion of an antibody constant region, which is typically that of a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized. For more details, see Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).

"Human antibodies" herein are antibodies comprising an amino acid sequence structure that corresponds to the amino acid sequence structure of an antibody produced by a human or human cell or derived from a non-human source (which utilizes a full complement of human antibodies or other human antibody coding sequences). This definition of a human antibody specifically excludes humanized antibodies that comprise a non-human antigen binding site. Such antibodies can be identified or prepared by a variety of techniques, including, but not limited to, production by transgenic animals (e.g., mice) that are capable, upon immunization, of producing human antibodies in the absence of endogenous immunoglobulin production (see, e.g., 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 U.S. Pat. Nos. 5,591,669, 5,589,369 and 5,545,807); selection from phage display libraries expressing human antibodies or human antibody fragments (see, e.g., McCafferty et al., Nature, 348:552-553 (1990); Johnson et al., Current Opinion in Structural 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); U.S. Patent Nos. 5,565,332 and 5,573,905); generated by in vitro activated B cells (see U.S. Patent Nos. 5,567,610 and 5,229,275); and isolated from hybridomas producing human antibodies.

"Multispecific antibodies" herein are antibodies that have binding specificity for at least two different epitopes. Exemplary multispecific antibodies can bind to both TfR and brain antigens. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab') ₂ bispecific antibodies). Engineered antibodies with two, three or more (e.g., four) functional antigen-binding sites are also contemplated (see, e.g., U.S. Application No. US 2002/0004587 A1, Miller et al.). Multispecific antibodies can be prepared as full-length antibodies or as antibody fragments.

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

" Affinity-modified variants " have one or more hypervariable regions or framework residues (e.g., parent chimeric, humanized, or human antibodies) of the parent antibody that are replaced, which change (increase or decrease) affinity. A convenient way to produce such substitution variants is to use phage display. In brief, several hypervariable region sites (e.g., 6-7 sites) are mutated to produce all possible amino substitutions at each site. The antibody variants thus produced are displayed as fusions (fusions) of the gene III product of the M13 packaged within each particle in a monovalent form by filamentous phage particles. The biological activity (e.g., binding affinity) of the phage-displayed variants is then screened. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that significantly contribute to antigen binding. Alternatively, or additionally, it may be advantageous to analyze the crystal structure of the antigen-antibody complex to identify the contact points between the antibody and its target. According to technology described herein, such contact residues and adjacent residues are candidates for substitution. After such variants are generated, the panel of variants is screened and antibodies with altered affinities can be selected for further development.

A "pH-sensitive antibody variant" is an antibody variant that has a binding affinity for a target antigen at a first pH that is different from its binding affinity for the target antigen at a different pH. As a non-limiting example, an anti-TfR antibody of the invention can be selected for or engineered to have pH-sensitive binding to TfR such that it binds to cell surface TfR in plasma at a desired low affinity (as described herein) at pH 7.4, but rapidly dissociates from TfR at a relatively low pH (pH 5.5-6.0) upon internalization into the endosomal compartment; such dissociation can protect the antibody from antigen-mediated clearance and increase the amount of antibody delivered to the CNS or recycled back across the BBB - in either case, the effective concentration of the antibody is increased relative to an anti-TfR antibody that does not contain such pH sensitivity (see, e.g., Chaparro-Riggers et al. J. Biol. Chem. 287(14): 11090-11097; Igawa et al. Nature Biotechnol. 28(11): 1203-1208). One of ordinary skill in the art can readily determine the ideal combination of affinity for TfR and conjugated compound at serum pH and at the pH of the endosomal compartment.

Antibodies can be conjugated to, for example, "heterologous molecules" herein to increase half-life or stability or otherwise improve the antibody. For example, antibodies can be connected to one of a variety of non-protein polymers (e.g., polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or a copolymer of polyethylene glycol and polypropylene glycol). Antibody fragments, such as Fab', connected to one or more PEG molecules are exemplary embodiments of the present invention. In another example, the heterologous molecule is a therapeutic compound or a visualizing agent (i.e., a detectable marker), and the antibody is used to transport this heterologous molecule across the BBB. Examples of heterologous molecules include, but are not limited to, chemical compounds, peptides, polymers, lipids, nucleic acids, and proteins.

The antibodies herein may be "glycosylation variants" so that any sugar (if any) connected to the Fc region is altered, modified in terms of presence/absence or modified in terms of type. For example, antibodies having a mature sugar structure lacking fucose coupled to the Fc region of an antibody are described in U.S. Patent Application No. US 2003/0157108 (Presta, L.). Also referring to US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). WO 2003/011878, Jean-Mairet et al. and U.S. Patent No. 6,602,684, Umana et al. mention antibodies having bisecting N-acetylglucosamine (GlcNAc) in the sugar connected to the Fc region of an antibody. Antibodies having at least one galactose residue in the oligosaccharide connected to the Fc region of an antibody are reported in WO 1997/30087, Patel et al. See also WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.) which relate to antibodies with altered carbohydrates attached to their Fc regions. See also US 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 297-299, where X cannot be proline) (e.g., by mutating the Asn of this sequence to any other amino acid, by placing Pro at position 298, or by modifying position 299 to any amino acid other than Ser or Thr) should eliminate glycosylation at this position (see, e.g., Fares AI-Ejeh et al., Clin. Cancer Res. (2007) 13:5519s-5527s; Imperiali 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).

The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and/or form structurally defined loops ("hypervariable loops") and/or contain antigen-contact residues ("antigen contacts"). Generally, antibodies comprise six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

(a) Hypervariable loops present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

(c) antigenic contacts present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); and

(d) a combination of (a), (b), and/or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

In one embodiment, the HVR residues comprise those identified in Figures 3A-D or 4A-D, Table 4 or Table 5, or elsewhere in this specification.

Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

"Framework" or "FR" residues refer to those variable domain residues other than the hypervariable region residues as defined herein. The FR of a variable domain is generally composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. In certain embodiments, one or more FR residues may be modified to modulate the stability of the antibody or to adjust the three-dimensional position of one or more HVRs of the antibody, for example, to enhance binding.

A "full-length antibody" is an antibody that comprises an antigen-binding variable region and a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

The terms "full length antibody," "intact antibody," and "intact antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody structure or has heavy chains that comprise an Fc region as defined herein.

A "naked antibody" is an antibody that is not conjugated to a heterologous moiety (such as a cytotoxic moiety or a radiolabel). The naked antibody can be present in a pharmaceutical formulation.

"Natural antibody" refers to naturally occurring immunoglobulin molecules with variable structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, which are composed of two identical light chains and two identical heavy chains, and the chains are bound by disulfide bonds. From N-terminal to C-terminal, each heavy chain has a variable region (VH), which is also referred to as a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from N-terminal to C-terminal, each light chain has a variable region (VL), which is also referred to as a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. Based on the amino acid sequence of its constant domains, the light chain of an antibody can be assigned to one of the two types referred to as kappa (κ) and lambda (λ).

Antibody "effector function" refers to those biological activities of an antibody that cause immune system activation other than complement pathway activation. Such activity is primarily found in the Fc region (native sequence Fc region or amino acid sequence variant Fc region) attributable to the antibody. Examples of antibody effector functions include, for example, Fc receptor binding and antibody-dependent cell-mediated cytotoxicity (ADCC). In one embodiment, the antibody herein substantially lacks effector function. In another embodiment, the antibody herein retains minimal effector function. Methods for modifying or eliminating effector function are well known in the art and include, but are not limited to, eliminating all or a portion of the Fc region that contributes to effector function (i.e., using an antibody or antibody fragment in a form lacking all or a portion of the Fc region (such as, but not limited to, Fab fragments, single chain antibodies, etc. as described herein and as known in the art); modifying the Fc region at one or more amino acid positions to eliminate effector function (Fc binding affects: positions 238, 239, 248, 249, 252, 254, 256, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 338, 339, 339, 331, 332, 333, 334, 335 , 298, 301, 303, 311, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 436, 437, 438 and 439; and modifying the glycosylation of the antibody (including but not limited to, producing the antibody in an environment that does not allow glycosylation by wild-type mammals, removing one or more carbohydrate groups from an already glycosylated antibody, and modifying the antibody at one or more amino acid positions to eliminate the ability of the antibody to be glycosylated at those positions, including but not limited to N297G and N297A and D265A).

Antibody "complement activation" function or the property of an antibody to allow or induce "activation of the complement pathway" are used interchangeably and refer to those biological activities of an antibody that participate in or stimulate the complement pathway of the immune system in a subject. Such activities include, for example, C1q binding and complement-dependent cytotoxicity (CDC) and can be regulated by both the Fc portion and the non-Fc portion of the antibody. Methods for modifying or eliminating 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 that causes complement activation (i.e., using antibodies or antibody fragments in a form lacking all or a portion of the Fc region (such as, but not limited to, Fab fragments, single-chain antibodies, etc. as described herein and as known in the art), or modifying the Fc region at one or more amino acid positions to eliminate or reduce interaction with complement components or the ability to activate complement components, such as positions 270, 322, 329, and 321, which are known to be involved in C1q binding), and modifying or eliminating a portion of the non-Fc region that causes complement activation (i.e., modifying the CH1 region at position 132 (see, e.g., Vidarte et al., (2001) J. Biol. Chem. 276(41):38217-38223)).

Depending on the amino acid sequence of the constant domain of its heavy chain, full-length antibodies can be assigned to different "classes". There are five major classes of full-length antibodies: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are known.

The term "recombinant antibody," as used herein, refers to an antibody (eg, a chimeric, humanized, or human antibody or antigen-binding fragment thereof) that is expressed by a recombinant host cell containing a nucleic acid encoding the antibody.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as that screened or selected for in the originally transformed cell are included herein. Examples of "host cells" for producing recombinant antibodies include: (1) mammalian cells, e.g., Chinese hamster ovary (CHO), COS, myeloma cells (including Y0 and NS0 cells), baby hamster kidney cells (BHK), Hela cells and Vero cells; (2) insect cells, e.g., sf9, sf21 and Tn5; (3) plant cells, e.g., plants belonging to the genus Nicotiana (e.g., Nicotiana tabacum); (4) yeast cells, e.g., those belonging to the genus Saccharomyces (e.g., Saccharomyces cerevisiae) or those belonging to the genus Aspergillus (e.g., Aspergillus niger); (5) bacterial cells, e.g., Escherichia coli cells or Bacillus subtilis cells, etc.

As used herein, "specific binding" or "specifically binds" means that the antibody selectively or preferentially binds to the antigen. Binding affinity is typically determined using standard assays such as Scatchard analysis or surface plasmon resonance technology (eg, using ).

An "antibody that binds to the same epitope as a reference antibody" refers to an antibody that blocks more than 50% of the binding of the reference antibody to its antigen in a competition assay, whereas the reference antibody blocks more than 50% of the binding of the antibody to its antigen in a competition assay. In one embodiment, the anti-BACE1 antibody that forms one of the bispecific or multispecific antibodies of the invention binds to the BACE1 epitope bound by YW412.8.31. Exemplary competition assays are provided herein.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioisotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C); C), chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleases; 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 various anti-tumor or anti-cancer agents disclosed herein.

An "effective amount" of an agent, such as a pharmaceutical agent, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Unless otherwise indicated, the numbering of the amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest (sequences of proteins of interest to immunology), 5th edition. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

The term "FcRn receptor" or "FcRn" as used herein refers to an Fc receptor ("n" indicating neonatal) known to be involved in the transfer of maternal IgGs to the fetus across the human or primate placenta or to the yolk sac (rabbit) to newborn animals via the small intestine. FcRn is also known to be involved in maintaining constant serum IgG levels by binding IgG molecules and recycling them into the serum. "FcRn binding region" or "FcRn receptor binding region" refers to the portion of an antibody that interacts with the FcRn receptor. Specific modifications in the FcRn binding region of an antibody increase the affinity of the antibody or fragment thereof for FcRn and also increase the in vivo half-life of the molecule. Amino acid substitutions at one or more of the following amino acid positions increase the interaction of an antibody with the FcRn receptor: 251, 256, 285, 290, 308, 314, 385, 389, 428, 434, and 436. Substitutions at the following positions also increase the interaction of the antibody with the FcRn receptor: 238, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitutions in (U.S. Pat. No. 7,371,826).

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to a label or a cytotoxic agent. Optionally, such conjugation is through a linker.

"Linker" as used herein is a structure that covalently or non-covalently connects an anti-TfR antibody to a heterologous molecule. In certain embodiments, the linker is a peptide. In other embodiments, the linker is a chemical linker.

A "label" is a marker that is coupled to the antibody herein and is used for detection or imaging. Examples of such labels include radiolabels, fluorophores, chromophores, or affinity tags. In one embodiment, the label is a radiolabel for medical imaging, such as tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, iron, and the like.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

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

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

"Isolated nucleic acid encoding an anti-TfR antibody" refers to one or more nucleic acid molecules that encode antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

"Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after the sequences are aligned (and, if necessary, introducing gaps) to obtain maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to achieve the purpose of determining the alignment of percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to obtain maximum alignment over the full length of the compared sequences. However, for this purpose, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The author of the ALIGN-2 sequence comparison computer program is Genentech, Inc., and the source code has been submitted to the U.S. Copyright Office (Washington D.C., 20559) with user documentation, and its U.S. copyright registration number is TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc. (South San Francisco, California) or can be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. The ALIGN-2 program sets all sequence alignment parameters and does not change.

In the case where ALIGN-2 is applied to amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (or in other words: a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

100 times the X/Y ratio

wherein X is the number of amino acid residues scored as identical matches using the sequence alignment program ALIGN-2 in that program's alignment of A and B, and wherein Y is the total number of amino acid residues in B. It will be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are derived using the ALIGN-2 computer program as described in the preceding paragraph.

The term "pharmaceutical formulation" refers to a preparation that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the formulation would be administered.

"Pharmaceutically acceptable carrier" refers to an ingredient other than the active ingredient in a pharmaceutical preparation that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, eliminating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and symptom relief or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of the disease or slow the progression of the disease.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). A single VH or VL domain can be sufficient to confer antigen binding specificity. In addition, VH or VL domains from antibodies that bind to a particular antigen can be used to isolate antibodies that bind to that antigen to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

2. Compositions and Methods

A. Preparation of anti-TfR antibodies and conjugates thereof

In one aspect, the present invention is based in part on anti-TfR antibodies that can be used to transport desired molecules across the BBB. In certain embodiments, antibodies that bind to human TfR are provided. In certain embodiments, antibodies that bind to both human TfR and primate TfR are provided. For example, the antibodies of the present invention can be used to diagnose or treat diseases that affect the brain and/or CNS.

A. Exemplary Anti-TfR Antibodies

In one aspect, the present invention provides isolated antibodies that bind to TfR. In certain embodiments, the anti-TfR antibodies of the present invention specifically bind to both human TfR and primate TfR. In certain such embodiments, the anti-TfR antibodies of the present invention do not inhibit the binding of transferrin to TfR. In certain such embodiments, the anti-TfR antibodies of the present invention bind to the apical domain of TfR. In certain other such embodiments, the anti-TfR antibodies of the present invention bind to the non-apical domain of TfR. In certain aspects, the anti-TfR antibodies can be used to transport one or more conjugated imaging or therapeutic compounds across the BBB.

In one aspect, the present invention provides an anti-TfR antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 29; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 30; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 31. In one aspect, the antibody comprises all six of the above HVR sequences. In another aspect, the antibody is clone 7A4, as shown in Figure 3A and Table 3.

In one aspect, the present invention provides an anti-TfR antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 30; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 36. In one aspect, the antibody comprises all six of the above HVR sequences. In another aspect, the antibody is clone 8A2, as shown in FIG3A and Table 3.

In one aspect, the present invention provides an anti-TfR antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 40; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 35; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 30; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 36. In one aspect, the antibody comprises all six of the above HVR sequences. In another aspect, the antibody is clone 15D2, as shown in FIG3A and Table 3.

In one aspect, the present invention provides an anti-TfR antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 43; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 44; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 41; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 30; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In one aspect, the antibody comprises all six of the above HVR sequences. In another aspect, the antibody is clone 10D11, as shown in Figure 3A and Table 3.

In one aspect, the present invention provides an anti-TfR antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 29; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 30; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 31. In one aspect, the antibody comprises all six of the above HVR sequences. In another aspect, the antibody is clone 7B10, as shown in Figure 3A and Table 3.

In one aspect, the present invention provides an anti-TfR antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 53; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 54; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 55; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 50; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 51; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 52. In one aspect, the antibody comprises all six of the above HVR sequences. In another aspect, the antibody is clone 15G11, as shown in Figure 3B and Table 3.

In one aspect, the present invention provides an anti-TfR antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 53; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 56; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 57; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 52. In one aspect, the antibody comprises all six of the above HVR sequences. In another aspect, the antibody is clone 16G5, as shown in Figure 3B and Table 3.

In one aspect, the present invention provides an anti-TfR antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 53; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 55; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 62. In one aspect, the antibody comprises all six of the above HVR sequences. In another aspect, the antibody is clone 13C3, as shown in Figure 3B and Table 3.

In one aspect, the present invention provides an anti-TfR antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 53; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 65; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 55; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 64; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 52. In one aspect, the antibody comprises all six of the above HVR sequences. In another aspect, the antibody is clone 16G4, as shown in Figure 3B and Table 3.

In one aspect, the present invention provides an anti-TfR antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 74; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 75; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 76; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 71; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 72; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 73. In one aspect, the antibody comprises all six of the above HVR sequences. In another aspect, the antibody is clone 16F6, as shown in Figure 3C and Table 3.

In one aspect, the present invention provides an anti-TfR antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 80; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 81; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 82; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 77; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 78; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 79. In one aspect, the antibody comprises all six of the above HVR sequences. In another aspect, the antibody is clone 7G7, as shown in Figure 3D and Table 3.

In one aspect, the present invention provides an anti-TfR antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 80; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 83; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 84; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 77; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 78; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 79. In one aspect, the antibody comprises all six of the above HVR sequences. In another aspect, the antibody is clone 4C2, as shown in Figure 3D and Table 3.

In one aspect, the present invention provides an anti-TfR antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 88; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 89; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 90; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 85; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 86; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 87. In one aspect, the antibody comprises all six of the above HVR sequences. In another aspect, the antibody is clone 1B12, as shown in Figure 3D and Table 3.

In one aspect, the present invention provides an anti-TfR antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 94; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 95; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 96; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 91; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 92; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 93. In one aspect, the antibody comprises all six of the above HVR sequences. In another aspect, the antibody is clone 13D4, as shown in Figure 3D and Table 3.

In one aspect, the present invention provides an anti-TfR antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 34; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 29; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 30; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 127. In one aspect, the antibody comprises all six of the above HVR sequences. In another aspect, the antibody is clone 7A4.v15, as shown in Figure 4B and Table 4.

The above clones fall into four complementarity groups with sequence similarity within HVRs. As shown in Table 3, the consensus sequence is easily derived from the antibody sequences provided for each HVR. As a non-limiting example, the consensus HVRs for Class I antibodies are as follows:

HVR-L1: Arg-Ala-Ser-Glu-Ser-Val-Asp-[Ser or Asp]-Tyr-Gly-[Asn or Pro]-Ser-Phe-Met-His (SEQ ID NO: 45);

HVR-L2: Arg-Ala-Ser-Asn-Leu-Glu-Ser (SEQ ID NO: 30);

HVR-L3: Gln-[Gln or His]-Ser-Asn-Glu-[Ala, Gly or Asp]-Pro-Pro-Thr (SEQ IDNO: 46);

HVR-H1: Asp-Tyr-[Ala or Gly]-Met-His (SEQ ID NO: 47);

HVR-H2: [Gly or Val]-Ile-Ser-[Thr, Phe or Pro]-Tyr-[Phe or Ser]-Gly-[Arg or Lys]-Thr-Asn-Tyr-[Asn or Ser]-Gln-[Lys or Asn]-Phe-[Lys or Met]-Gly (SEQ ID NO: 48);

HVR-H3: Gly-Leu-Ser-Gly-Asn-[Tyr or Phe]-Val-[Met or Val]-Asp-[Tyr or Phe] (SEQ ID NO: 49). (See Table 4.) The consensus sequences for classes II and IV are also provided in Table 3.

In one aspect, the present invention provides an anti-TfR antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 47; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 48; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 49; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 45; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 30; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 46. In one aspect, the antibody comprises all six of the above HVR sequences.

In one aspect, the present invention provides an anti-TfR antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 53; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 69; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 70; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 66; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 67; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 68. In one aspect, the antibody comprises all six of the above HVR sequences.

In one aspect, the present invention provides an anti-TfR antibody comprising at least one, two, three, four, five or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 101; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 97; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 98; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 99. In one aspect, the antibody comprises all six of the above HVR sequences.

In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences of any of the aforementioned antibodies. In one embodiment, the antibody comprises the HVR-H3 sequence of any of the aforementioned antibodies. In another embodiment, the antibody comprises the HVR-H3 and HVR-L3 sequences of any of the aforementioned antibodies. In another embodiment, the antibody comprises the HVR-H3, HVR-L3, and HVR-H2 sequences of any of the aforementioned antibodies. In another embodiment, the antibody comprises the HVR-H1, HVR-H2, and HVR-H3 sequences of any of the aforementioned antibodies. In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences of any of the aforementioned antibodies. In one embodiment, the antibody comprises the HVR-L1, HVR-L2, and HVR-L3 sequences of any of the aforementioned antibodies.

In another aspect, an antibody of the invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from the HVR-H1, HVR-H2, and HVR-H3 sequences of any one of the aforementioned antibodies; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from the HVR-L1, HVR-L2, and HVR-L3 sequences of any one of the aforementioned antibodies.

In any of the foregoing embodiments, the anti-TfR antibody is humanized. In one embodiment, the anti-TfR antibody comprises HVRs as in any of the above embodiments, and further comprises an acceptor human framework, for example, a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-TfR antibody comprises HVRs as in any of the above embodiments, and further comprises a VH or VL containing one or more amino acid substitutions in one or more FR regions. According to Example 2 herein, the applicant performed alanine scanning on certain antibodies selected from those described above, and determined that, despite the amino acid modifications at the selected FR positions, similar or improved binding was obtained. As shown in Figures 6-1 and 6-2 and Example 2 herein, for the class I-III antibody group, the antibodies retain affinity and binding specificity in variant forms having modifications at one or more residues in the FR. For example, for antibody 15G11, positions 43 and 48 in the light chain FR2, position 48 in the heavy chain FR2, and positions 67, 69, 71, and 73 in the heavy chain FR3 can be modified as shown in Figures 6-1 and 6-2, and the resulting antibodies still retain specificity and strong binding affinity for human/primate TfR. In another example, for antibody 7A4, positions 58 and 68 in the light chain FR3, position 24 in the heavy chain FR1, and position 71 in the heavy chain FR3 can be modified as shown in Figures 6-1 and 6-2, and the resulting antibodies still retain specificity and strong binding affinity for human/primate TfR. In a third example, for antibody 16F6, positions 43 and 44 in the light chain FR2 and positions 71 and 78 in the heavy chain FR3 can be modified as shown in Figures 6-1 and 6-2, and the resulting antibodies still retain specificity and strong binding affinity for human/primate TfR. In another aspect, the anti-TfR antibody comprises a heavy chain variable domain (VH) sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 7-10, 15-18, 20, 25-28, 108, 114, 120 and 126. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity relative to the reference sequence comprises substitutions (e.g., conservative substitutions), insertions or deletions, but the anti-TfR antibody comprising the sequence retains the ability to bind to TfR. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in any one of SEQ ID NOs: 7-10, 15-18, 20, 25-28, 108, 114, 120 and 126. In certain embodiments, the substitutions, insertions or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-TfR antibody comprises the VH sequence of any one of SEQ ID NOs: 7-10, 15-18, 20, 25-28, 108, 114, 120 and 126, including post-translational modifications of said sequence. In a particular embodiment, the VH of a particular antibody comprises one, two or three HVRs selected from the following: the HVRs listed above and in Table 3 or 4 for said particular antibody. The VH sequences for the antibodies of the present invention are shown in Figures 3 and 4 herein.

In another aspect, an anti-TfR antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 4-6, 11-14, 19, 21-24, 105, 111, 117 and 123. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity relative to the reference sequence comprises substitutions (e.g., conservative substitutions), insertions or deletions, but the anti-TfR antibody comprising the sequence retains the ability to bind to TfR. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in any one of SEQ ID NOs: 4-6, 11-14, 19, 21-24, 105, 111, 117 and 123. In certain embodiments, the substitutions, insertions or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-TfR antibody comprises the VL sequence of any one of SEQ ID NOs: 4-6, 11-14, 19, 21-24, 105, 111, 117, and 123, including post-translational modifications of said sequence. In a particular embodiment, the VL of a specific antibody comprises one, two or three HVRs selected from the following: the HVRs listed above and in Table 4 or 5 for the specific antibodies. The VL sequences for the antibodies of the present invention are shown in Figures 3 and 4 herein.

In another aspect, an anti-TfR antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the following VL and VH sequences, respectively: SEQ ID NOs: 4 and 7; 5 and 8; 5 and 9; 6 and 10; 4 and 7; 11 and 15; 12 and 16; 13 and 17; 14 and 18; 19 and 20; 21 and 25; 22 and 26; 23 and 27; 24 and 28; 105 and 108; 111 and 114; 117 and 120; and 123 and 126, including post-translational modifications of said sequences.

In another aspect, the present invention provides antibodies that bind to the same epitope as the anti-TfR antibodies provided herein. For example, in certain embodiments, antibodies are provided that bind to the same epitope as an anti-TfR antibody comprising the VL and VH sequences of SEQ ID NOs: 4 and 7; 5 and 8; 5 and 9; 6 and 10; 4 and 7; 11 and 15; 12 and 16; 13 and 17; 14 and 18; 19 and 20; 21 and 25; 22 and 26; 23 and 27; 24 and 28; 105 and 108; 111 and 114; 117 and 120; or 123 and 126, respectively. In one aspect, the antibody competes for binding to TfR with any of the antibodies in Class I (i.e., clones 7A4, 8A2, 15D2, 10D11 or 7B10 or affinity matured versions of any of these antibodies). In another aspect, the antibody competes for binding to TfR with any one of the antibodies in class II (i.e., clones 15G11, 16G5, 13C3 or 16G or affinity matured versions of any of these antibodies). In another aspect, the antibody competes for binding to TfR with clone 16F6 or affinity matured versions thereof. In another aspect, the antibody competes for binding to TfR with any one of the antibodies in class IV (i.e., clones 7G7, 4C2, 1B12 or 13D4 or affinity matured versions thereof).

In another aspect of the invention, the anti-TfR antibody described in any of the above embodiments is a monoclonal antibody, including chimeric, humanized or human antibodies. In one embodiment, the anti-TfR antibody is an antibody fragment, e.g., Fv, Fab, Fab', scFv, diabody or F(ab') ₂ fragment. In another embodiment, the antibody is a full-length antibody, e.g., a complete IgG1, IgG2, IgG3 or IgG4 antibody or other antibody classes or isotypes described herein.

In another aspect, the anti-TfR antibody of any of the above embodiments may be combined with any of the features described in aspects 1-7 below (alone or in combination):

1. Antibody affinity

In certain embodiments, the antibodies provided herein have a dissociation constant (Kd) ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 ⁻⁸ M or less, e.g., 10 ⁻⁸ M to 10 ⁻¹³ M, e.g., ^{10 −9 M} to 10 ⁻¹³ M).

In certain aspects of the invention, the "low affinity" anti-TfR antibodies of the invention are selected based on, for example, the results of Atwal et al., Sci. Transl. Med. 3, 84ra43 (2011) and Yu et al., Sci. Transl. Med. 25 May 2011: Vol. 3, Issue 84, p. 84ra44 of Example 5, which show that such low affinity antibodies against TfR exhibit improved CNS (e.g., brain) uptake and/or persistence in the brain/CNS. To identify such low affinity antibodies, a variety of assays for measuring antibody affinity are available, including, but not limited to, Scatchard assays and surface plasmon resonance technology (e.g., using ). According to one embodiment of the invention, the affinity of the antibody to human or primate TfR is from about 5 nM, or from about 20 nM, or from about 100 nM, to about 50 μM, or to about 30 μM, or to about 10 μM, or to about 1 μM, or to about 500 nM. Thus, the affinity can be in the range of about 5 nM to about 50 μM, or in the range of about 20 nM to about 30 μm, or in the range of about 30 nM to about 30 μm, or in the range of about 50 nM to about 1 μM, or in the range of about 100 nM to about 500 nM, for example, as measured by a Scatchard assay or otherwise. In another embodiment of the invention, the dissociation half-life of the antibody to TfR is less than 1 minute, less than 2 minutes, less than 3 minutes, less than 4 minutes, less than 5 minutes, or less than 10 minutes to about 20 minutes, or to about 30 minutes, as measured by a competitive binding assay or otherwise.

Thus, the present invention provides a method for preparing an antibody that can be used to transport a neurological disease drug across the blood-brain barrier, the method comprising selecting an antibody from a panel of antibodies against TfR (because its affinity for TfR is in the range of from about 5 nM, or from about 20 nM, or from about 100 nM, to about 50 μM, or to about 30 μM, or to about 10 μM, or to about 1 μM, or to about 500 mM). Thus, the affinity can be in the range of about 5 nM to about 50 μM, or in the range of about 20 nM to about 30 μm, or in the range of about 30 nM to about 30 μm, or in the range of about 50 nM to about 1 μM, or in the range of about 100 nM to about 500 nM, for example, as measured by Scatchard analysis or. As will be understood by one of ordinary skill in the art, conjugating a heterologous molecule/compound to an antibody will generally reduce the affinity of the antibody for its target due to, for example, steric hindrance or even due to the elimination of one binding arm (if the antibody is made multispecific and has one or more arms that bind to an antigen different from the original target of the antibody). In one embodiment, a low affinity antibody of the invention that is specific for TfR conjugated to anti-BACE1 has a Kd for TfR of about 30 nM as measured by BIACORE. In another embodiment, a low affinity antibody of the invention that is specific for TfR conjugated to BACE1 has a Kd for TfR of about 600 nM as measured by BIACORE. In another embodiment, a low affinity antibody of the invention that is specific for TfR conjugated to BACE1 has a Kd for TfR of about 20 μM as measured by BIACORE. In another embodiment, a low affinity antibody of the invention specific for TfR conjugated to BACE1 has a Kd for TfR of about 30 μM as measured by BIACORE.

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with a Fab form of the antibody of interest and its antigen. For example, the solution binding affinity of a Fab for an antigen is measured by equilibrating the Fab with a minimal concentration of ( ¹²⁵ I)-labeled antigen in the presence of a titration series of unlabeled antigen, followed by capturing the bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293: 865-881 (1999)). To determine the conditions for the assay, multiwell plates (Thermo Scientific) were coated overnight with 5 μg/ml of a capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and subsequently blocked with 2% (w/v) bovine serum albumin in PBS at room temperature (approximately 23° C.) for 2-5 hours. In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [ ¹²⁵ I]-antigen is mixed with a serial dilution of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody, Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation can be continued for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 in PBS. When the plate has dried, 150 μl/well of scintillant (MICROSCINT-20 ^™ ; Packard) is added and the plate is counted for 10 minutes on a TOPCOUNT ^™ gamma counter (Packard). A concentration of each Fab that gave less than or equal to 20% of maximal binding was chosen for use in competitive binding assays.

In one aspect, RIA is a Scatchard analysis. For example, the target anti-TfR antibody can be iodinated using the lactoperoxidase method (Bennett and Horuk, Methods in Enzymology 288 pg.134-148 (1997)). Radiolabeled anti-TfR antibodies are purified using a NAP-5 column by gel filtration to separate them from free ¹²⁵ I-Na, and their specific activity is measured. 50 μL of a competition reaction mixture containing a fixed concentration of iodinated antibody and a decreasing concentration of serially diluted unlabeled antibody is placed in a 96-well plate. Cells transiently expressing TfR are cultured in growth medium at 37°C in 5% _CO2 , and the growth medium consists of Dulbecco's modified eagle's medium (DMEM) (Genentech) supplemented with 10% FBS, 2mM L-glutamine and 1× penicillin-streptomycin. Cells were detached from the culture dishes using Sigma Cell Dissociation Solution and washed with binding buffer (DMEM containing 1% bovine serum albumin, 50 mM HEPES, pH 7.2, and 0.2% sodium azide). The washed cells were added to a 96-well plate containing a 50-μL competition reaction mixture at an approximate density of 200,000 cells/0.2 mL binding buffer. The final concentration of unlabeled antibody in the competition reaction containing cells was varied, starting at 1000 nM and then decreasing by 1:2 dilution to 10 concentrations, including a sample with zero addition and only buffer. The competition reaction containing cells was measured in triplicate for each concentration of unlabeled antibody. The competition reaction containing cells was incubated at room temperature for 2 hours. After the 2-hour incubation, the competition reaction solution was transferred to a filter plate and washed four times with binding buffer to separate free and bound iodinated antibodies. The filters were counted by a gamma counter and the binding data were evaluated using the fitting algorithm of Munson and Rodbard (1980) to determine the binding affinity of the antibody.

An exemplary analysis using the compositions of the present invention can be performed as follows. Kd was measured using a surface plasmon resonance assay using a BIAcore-2000 (BIAcore, Inc., Piscataway, NJ) at 25°C using an anti-human Fc kit (BiAcore Inc., Piscataway, NJ). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) were activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxy-succinimide (NHS) according to the supplier's instructions. Anti-human Fc antibodies were diluted to 50 μg/ml with 10 mM sodium acetate, pH 4.0, and then injected at a flow rate of 5 μl/min to obtain approximately 10,000 response units (RU) of coupled protein. Following the injection of the antibody, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, monospecific or multispecific anti-TfR antibody variants were injected into HBS-P to reach approximately 220 RU, and then two-fold serial dilutions of MuTfR-His (0.61 nM to 157 nM) were injected into HBS-P at a flow rate of approximately 30 μl/min at 25° C. The association rate (k on) and dissociation rate (k off) were calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (Evaluation Software version 3.2). The equilibrium dissociation constant (K d ) was calculated as the ratio k off / k on. See, e.g., Chen et al., J. Mol. Biol. 293: 865-881 (1999).

According to another embodiment, Kd is measured using a surface plasmon resonance assay using a BIAcore-2000 apparatus (BIAcore, Inc., Piscataway, NJ) at 25°C using an anti-human Fc kit (BiAcore Inc., Piscataway, NJ). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxy-succinimide (NHS) according to the supplier's instructions. Anti-human Fc antibody was diluted to 50 μg/ml with 10 mM sodium acetate, pH 4.0, and then injected at a flow rate of 5 μl/min to obtain approximately 10,000 response units (RU) of coupled protein. Following the injection of the antibody, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, anti-TfR antibody variants were injected into HBS-P to reach approximately 220 RU, and then two-fold serial dilutions of TfR-His (0.61 nM to 157 nM) were injected into HBS-P at a flow rate of approximately 30 μl/min at 25° C. The association rate (k on) and dissociation rate (k off) were calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (Evaluation Software version 3.2). The equilibrium dissociation constant (K d ) was calculated as the ratio k off / k on. See, e.g., Chen et al., J. Mol. Biol. 293: 865-881 (1999).

Several methods are known for determining the IC50 of a given compound; a common method is to perform a competition binding assay, such as described herein. Generally, a high IC50 indicates that more antibody is required to inhibit binding of a known ligand, and therefore, the antibody has a relatively low affinity for the ligand. Conversely, a low IC50 indicates that less antibody is required to inhibit binding of a known ligand, and therefore, the antibody has a relatively high affinity for the ligand.

An exemplary competitive ELISA assay for measuring IC50 is one in which increasing concentrations of anti-TfR or anti-TfR/brain antigen (i.e., anti-TfR/BACE1, anti-TfR/Aβ, etc.) variant antibodies are used to compete with biotinylated known anti-TfR antibodies for binding to TfR. The anti-TfR competition ELISA was performed overnight at 4°C in Maxisorp plates (Neptune, N.J.) coated with 2.5 μg/ml purified mouse TfR extracellular domain (in PBS). The plates were washed with PBS/0.05% Tween 20 and blocked with Superblock blocking buffer (Thermo Scientific, Hudson, NH) in PBS. Each individual anti-TfR or anti-TfR/brain antigen (i.e., anti-TfR/BACE1 or anti-TfR/Aβ) titration (1:3 serial dilution) was combined with biotinylated anti-TfR (0.5 nM final concentration) and added to the plate for 1 hour at room temperature. The plate was washed with PBS/0.05% Tween 20, and HRP-streptavidin (Southern Biotech, Birmingham) was added to the plate and incubated for 1 hour at room temperature. The plate was washed with PBS/0.05% Tween 20, and biotinylated anti-TfR antibodies bound to the plate were detected using TMB substrate (BioFX Laboratories, Owings Mills).

2. Antibody fragments

In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv and scFv fragments, as well as other fragments described below. For a review of specific antibody fragments, see Hudson et al., Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, for example, Pluckthün, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab') ₂ fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see US Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, e.g., EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

A single domain antibody is an antibody fragment comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl).

Antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage), as described herein.

3. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. Chimeric antibodies of interest herein include "primatized" antibodies, which comprise variable domain antigen-binding sequences derived from a non-human primate (e.g., an Old World Monkey, such as a baboon, rhesus, or cynomolgus monkey) and human constant region sequences (U.S. Pat. No. 5,693,780). In another example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while maintaining the specificity and affinity of the parent non-human antibody. Generally speaking, humanized antibodies comprise one or more variable domains, wherein HVRs, such as CDRs (or portions thereof) are derived from non-human antibodies, and FRs (or portions thereof) are derived from human antibody sequences. Humanized antibodies optionally also will comprise at least a portion of a human constant region. In some embodiments, some FR residues in humanized antibodies are replaced with corresponding residues from non-human antibodies (such as antibodies from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further in, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "surface remodeling"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection" method of FR shuffling).

Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (e.g., see Sims et al. J. Immunol. 151: 2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (e.g., see Carter et al. Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); and Presta et al. J. Immunol., 151: 2623 (1993)). 3)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be prepared using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified so that it stimulates the production of intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. These animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or are present extrachromosomally or randomly integrated into the animal's chromosomes. In these transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE ^™ technology; U.S. Patent No. 5,770,429 describing the technology; U.S. Patent No. 7,041,870 describing the KM technology, and U.S. Patent Application Publication No. US 2007/0061900 describing the technology. The human variable regions of the intact antibodies produced by these animals can be further modified, for example, by combining them with different human constant regions.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991).) Human antibodies produced by human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Other methods include those described in, for example, U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies by hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005), and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

It is also possible to produce human antibodies by separating the Fv clone variable domain sequences selected from the phage display library derived from people. These variable domain sequences can then be combined with desired human constant domains. The technology of selecting human antibodies from the antibody library is described below.

5. Antibodies from the library

Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies with the desired activity. For example, a variety of methods are known in the art for generating phage display libraries and screening these libraries for antibodies with the desired binding characteristics. These methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001), and further in, for example, McCafferty et al., Nature 348: 552-554; Clackso et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248: 161-175 (Lo eds., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

In certain phage display methods, the VH and VL repertoires are cloned separately by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments in the form of single-chain Fv (scFv) fragments or Fab fragments. Libraries from immune sources can provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, natural repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a variety of non-self antigens as well as self antigens without any immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, natural libraries can also be made synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 regions and effecting in vitro rearrangement, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificity for at least two different sites. In certain embodiments, one binding specificity is for TfR and the other is for any other antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of TfR. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing TfR. Bispecific antibodies can be made into full-length antibodies or antibody fragment forms.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 93/08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be made by engineering electrostatic forces for making antibody Fc-heterodimers (WO 93/08829, and Traunecker et al., EMBO J. 10:3655 (1991)). 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980, and Brennan et al. Science, 229:81 (1985)); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to make bispecific Antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and trispecific antibodies as described, e.g., in Tutt et al., J. Immunol. 147:60 (1991).

The antibodies or fragments herein also include "dual-acting Fabs" or "DAFs" that comprise antigen binding sites that bind to TfR as well as another, different antigen (see, eg, US 2008/0069820).

According to one embodiment of the present invention, "coupling" is achieved by generating a multispecific antibody (e.g., a bispecific antibody). A multispecific antibody is a monoclonal antibody that has binding specificity for at least two different antigens or epitopes. In one embodiment, the multispecific antibody comprises a first antigen binding site that binds to TfR and a second antigen binding site that binds to a brain antigen, such as β-secretase 1 (BACE1) or Aβ, and other brain antigens disclosed herein.

An exemplary brain antigen bound by such a multispecific/bispecific antibody is BACE1, and an exemplary antibody that binds thereto is the YW412.8.31 antibody in Figures 16A-B herein.

In another embodiment, the brain antigen is Aβ, exemplary such antibodies are disclosed in WO2007068412, WO2008011348, WO20080156622, and WO2008156621 (which are expressly incorporated herein by reference), where exemplary Aβ antibodies include the IgG4MABT5102A antibody comprising the heavy and light chain amino acid sequences in Figures 11A and 11B, respectively.

Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829 and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic directing effects for preparing antibody Fc-heterodimers (WO 93/08829 and Traunecker et al., EMBO J. 10: 3655 (1991)). 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to make bispecific antibodies and trispecific antibodies prepared using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)) and as described, e.g., in Tutt et al., J. Immunol. 147:60 (1991).

Also included herein are engineered antibodies with more than three functional antigen-binding sites, including "Octopus antibodies" or "dual-variable domain immunoglobulins" (DVDs) (see, e.g., US 2006/0025576A1, and Wu et al., Nature Biotechnology (2007)).

7. Antibody variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are encompassed. For example, it may be necessary to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. These modifications include, for example, deletions and/or insertions of residues within the antibody amino acid sequence and/or replacements thereof. Any combination of deletion, insertion, and replacement can be performed to obtain the final construct, with the proviso that the final construct has the desired characteristics, such as antigen binding.

a) Substitution, insertion and deletion variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. The relevant sites for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 2 under the heading "Preferred Substitutions." More substantial changes are provided under the heading "Exemplary Substitutions" in Table 2 and are further described below with respect to amino acid side chain types. Amino acid substitutions can be introduced into related antibodies and screened for products with desired activity, such as maintained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

Table 2

Amino acids can be grouped according to common side chain properties:

(1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

(2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

(3) Acidic: Asp, Glu;

(4) Basic: His, Lys, Arg;

(5) Residues that affect chain orientation: Gly, Pro;

(6) Aromaticity: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another.

One type of substitutional variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). In general, certain biological properties of the resulting variants selected for further study are changed (e.g., improved) relative to the parent antibody (e.g., increased affinity, reduced immunogenicity) and/or will substantially retain certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which can be conveniently produced, for example, using affinity maturation techniques based on phage display, such as those described herein. In short, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for a specific biological activity (e.g., binding affinity).

Changes (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. These changes can be made in HVR "hotspots," i.e., residues encoded by codons that undergo high frequency mutation during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)); and/or in residues that contact the antigen, where the resulting variant VH or VL is tested for binding affinity. Affinity maturation by constructing and reselecting from a secondary library has been described, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods, such as error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis. A secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomly selected. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. Specifically, CDR-H3 and CDR-L3 are typically targeted.

In certain embodiments, substitutions, insertions or deletions may be made within one or more HVRs, as long as these changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made in HVRs. For example, these changes may be outside the antigen contact residues in the HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or contains no more than one, two or three amino acid substitutions.

A method suitable for identifying residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, residues or groups of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Additional substitutions can be introduced at amino acid positions where the initial substitutions showed functional sensitivity. Alternatively or additionally, a crystal structure of the antigen-antibody complex is used to identify contact points between the antibody and the antigen. These contact residues and neighboring residues can be targeted or eliminated as substitution candidates. The variants can be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing more than one hundred residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of the antibody molecule include fusions of the N- or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

b) Glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibodies are glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

If the antibody comprises an Fc region, the carbohydrates attached thereto may be altered. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, generally linked via an N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH 15: 26-32 (1997). Oligosaccharides may include a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose linked to the GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention may be modified to produce antibody variants with certain improved properties.

In one embodiment, antibody variants are provided that lack fucose glycostructures attached (directly or indirectly) to the Fc region. For example, the amount of fucose in the antibody can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose at Asn297 within the sugar chain relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid, and high-mannose structures) as measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008/077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (Eu numbering of Fc region residues); however, due to minor sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. These fucosylation variants may have improved ADCC function. See, e.g., U.S. Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Okazaki et al., J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., particularly Example 11); and gene knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, gene knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2004/056312 A1, Adams et al., particularly Example 11). 2003/085107).

Further provided are antibody variants having bisected oligosaccharides, for example, wherein the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. These antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of these antibody variants are described, for example, in WO 2003/011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. These antibody variants may have improved CDC function. These antibody variants are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc region variants

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

In certain embodiments, the present invention encompasses antibody variants that possess some, but not all, effector functions, making them ideal candidates for certain applications where the in vivo half-life of the antibody is important but certain effector functions (e.g., complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/exhaustion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity) but retains FcRn binding ability. The primary cell mediating ADCC, NK cells, expresses only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of related molecules are described in U.S. Pat. Nos. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, e.g., Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be employed (see, e.g., the ACTI ^™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA) and the CvtoTox non-radioactive cytotoxicity assay (Promega, Madison, WI)). Suitable effector cells for these assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the relevant molecules can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95: 652-656 (1998). C1q binding assays can also be performed to demonstrate that the antibody is unable to bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

Examples of antibodies with reduced effector function include those with one or more substitutions in the Fc region residues 238, 265, 269, 270, 297, 327, and 329 ( U.S. Pat. No. 6,737,056 ). These Fc mutants include those with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine ( U.S. Pat. No. 7,332,581 ).

Certain antibody variants with improved or reduced binding to FcRs are described (see, e.g., U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that increase ADCC, such as substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).

In some embodiments, alterations are made in the Fc region that result in altered (i.e., by increasing or decreasing) C1q binding and/or complement-dependent cytotoxicity (CDC), as described, e.g., in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

US 2005/0014934A1 (Hinto et al.) describes antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). These antibodies comprise an Fc region with one or more substitutions that improve binding of the Fc region to FcRn. Such non-limiting Fc variants include those having substitutions at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, e.g., substitution of Fc region residue 434 ( U.S. Pat. No. 7,371,826 ).

See also Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351 for other examples of Fc region variants.

d) Cysteine-engineered antibody variants

In certain embodiments, it may be desirable to produce antibodies engineered with cysteine, such as "thioMAbs," in which one or more residues of an antibody are replaced with cysteine residues. In a specific embodiment, the replaced residues occur at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are located at accessible sites of the antibody and can be used to bind the antibody to other moieties, such as drug moieties or linker-drug moieties, to produce immunoconjugates as further described herein. In certain embodiments, any one or more of the following residues may be replaced with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies can be produced, for example, as described in U.S. Patent No. 7,521,541.

e) Antibody derivatives

In certain embodiments, the antibodies provided herein may be further modified to contain other non-proteinaceous moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxanetriane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, it may be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used as a therapy for a given condition, etc.

In another embodiment, a conjugate of an antibody with a non-protein moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, including, but not limited to, wavelengths that do not damage normal cells but heat the non-protein moiety to a temperature that kills cells in the vicinity of the antibody-non-protein moiety.

B. Recombinant Methods and Compositions

Antibodies can be produced using the recombinant methods and compositions described, for example, in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-TfR antibody described herein is provided. Such nucleic acids can encode an amino acid sequence comprising the VL of the antibody and/or an amino acid sequence comprising its VH (e.g., a light chain and/or a heavy chain of the antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., transformed with the following): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method for preparing an anti-TfR antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium).

To recombinantly produce an anti-TfR antibody, nucleic acid encoding the antibody (e.g., the antibody described above) is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli). After expression, the antibodies can be separated from the bacterial cell paste in the soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).

Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES ^™ technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney line (293 or 293 cells, as described, for example, in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather et al., Annals of [0015] Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR ^- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), and myeloma cell lines such as Y0, NS0, and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

C. Determination

The anti-TfR antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.

1. Binding Assays and Other Assays

There are a variety of techniques that can be used to determine the binding of antibodies to TfR. One such assay is an enzyme-linked immunosorbent assay (ELISA) for verifying the ability to bind to human TfR (and brain antigens). According to this assay, a plate coated with an antigen (e.g., recombinant TfR) is incubated with a sample containing an anti-TfR antibody, and the binding of the antigen to the antigen of interest is determined.

In one aspect, the antigen binding activity of the antibodies of the invention is tested, for example, by known methods such as ELISA, Western blot, and the like.

In another aspect, competition assays can be used to identify antibodies that compete with any of the antibodies described herein for binding to TfR. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) that is bound by any of the antibodies described herein, more specifically, to any of the epitopes specifically bound by Class I, Class II, Class III, or Class IV antibodies described herein (e.g., see Example 1 and Table 4). Detailed exemplary methods for locating which epitope an antibody binds to are provided in Morris (1996) "Epitope Mapping Protocols," Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ).

In an exemplary competition assay, immobilized TfR is incubated in a solution comprising a first labeled antibody (e.g., one or more of the antibodies disclosed herein) that binds to TfR and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to TfR. The second antibody can be present in a hybridoma supernatant. As a control, immobilized TfR is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to TfR, excess unbound antibody is removed and the amount of label bound to the immobilized TfR is measured. If the amount of label bound to the immobilized TfR in the test sample is significantly reduced relative to the control sample, the second antibody is competing with the first antibody for binding to TfR. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

2. Activity Assay

In one aspect, assays are provided for identifying anti-TfR antibodies with biological activity. The biological activity can include, for example, transporting compounds associated/conjugated to the antibody across the BBB into the brain and/or CNS. Antibodies with such biological activity in vivo and/or in vitro are also provided.

In certain embodiments, the antibodies of the invention are tested for such biological activities.

D. Immunoconjugates

The present invention also provides immunoconjugates comprising an anti-TfR antibody herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, the anti-TfR antibodies herein are conjugated to a neurological disease drug, chemotherapeutic agent, and/or imaging agent to more efficiently transport the drug, chemotherapeutic agent, and/or imaging agent across the BBB.

Covalent conjugation can be direct or achieved through a linker. In certain embodiments, direct conjugation is achieved by constructing a protein fusion (i.e., by genetic fusion of two genes encoding anti-TfR antibodies and, for example, neurological disease drugs and expressed as a single protein). In certain embodiments, direct conjugation is achieved by forming a covalent bond between a reactive group on one of the two parts of an anti-TfR antibody and, for example, a corresponding group or receptor on a neurological drug. In certain embodiments, direct conjugation is achieved by modifying (i.e., genetically modified) one of the two molecules to be conjugated to comprise a reactive group (as a non-limiting example, a sulfhydryl or carboxyl group), which forms a covalent coupling with another molecule to be conjugated under suitable 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 into, for example, an anti-TfR antibody and forming a disulfide bond with, for example, a neurological drug. Methods for covalently conjugating nucleic acids to proteins are also known in the art (ie, photocrosslinking, see, eg, Zatsepin et al., Russ. Chem. Rev. 74:77-95 (2005)).

Non-covalent conjugation can be through any non-covalent attachment means, including hydrophobic bonds, ionic bonds, electrostatic interactions, etc., as readily understood by those of ordinary skill in the art.

Conjugation can also be performed using a variety of linkers. For example, anti-TfR antibodies and neurological drugs can be conjugated using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See WO 94/11026. Peptide linkers consisting of one to twenty amino acids linked by peptide bonds can also be used. In certain such embodiments, the amino acids are selected from the twenty naturally occurring amino acids. In certain other such embodiments, one or more amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. The linker can be a "cleavable linker" that facilitates release of the neurological drug after delivery to the brain. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020).

The present invention expressly contemplates, but is not limited to, conjugates prepared using cross-linking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A.).

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including, but not limited to, maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235). B1); auristatin, such as the monomethylauristatin moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin; calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. Res. 58: 2925-2928 (1998)); anthracyclines, such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13: 477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16: 358-362 (2006); Torgov et al., Bioconj. Chem. 16: 717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97: 829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vindesine; taxanes, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis toxin, officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to prepare radioconjugates. Examples include At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioisotopes of Lu. When a radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, such as tc 99m or I 123 or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 and iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

E. Methods and compositions for diagnosis and detection

In certain embodiments, any of the anti-TfR antibodies provided herein can be used to detect the presence of TfR in a biological sample. The term "detection" as used herein includes quantitative or qualitative detection. In certain embodiments, the biological sample includes cells or tissues, such as blood (i.e., immature red blood cells), CSF, and tissues comprising the BBB.

In one embodiment, an anti-TfR antibody for use in a diagnostic or detection method is provided. In another aspect, a method for detecting the presence of TfR in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with an anti-TfR antibody as described herein under conditions that allow the anti-TfR antibody to bind to TfR, and detecting whether a complex is formed between the anti-TfR antibody and TfR. The method can be an in vitro or in vivo method. In one embodiment, an anti-TfR antibody is used to select a subject suitable for treatment with an anti-TfR antibody, for example, where TfR is a biomarker for selecting a patient.

Since TfR is expressed in reticulocytes and can therefore be detected using any of the antibodies of the invention, exemplary conditions that can be diagnosed using the antibodies of the invention include conditions involving immature red blood cells. Such conditions include anemia and other conditions caused by decreased reticulocyte levels or congenital polycythemia or neoplastic polycythemia vera, in which elevated red blood cell counts due to, for example, excessive proliferation of reticulocytes lead to thickening of the blood and associated physiological symptoms.

In certain embodiments, labeled anti-TfR antibodies are provided. Labels include, but are not limited to, directly detected labels or moieties (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as indirectly detected moieties, such as enzymes or ligands, e.g., by enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferase, for example, firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases, for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, plus enzymes that utilize hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, phage labels, stable free radicals, and the like.

In one embodiment, the intact antibody lacks effector function. In another embodiment, the intact antibody has reduced effector function. In another embodiment, the intact antibody is engineered to have reduced effector function. In one aspect, the antibody is a Fab. In another aspect, the antibody has one or more Fc mutations that reduce or eliminate effector function. In another aspect, the antibody has modified glycosylation, for example, due to production of the antibody in a system lacking normal human glycosylation enzymes. In another aspect, the Ig backbone is modified to a backbone that naturally has limited or no effector function.

There are a variety of techniques that can be used to determine the binding of antibodies to TfR. One such assay is an enzyme-linked immunosorbent assay (ELISA) for verifying the ability to bind to human TfR (and brain antigens). According to this assay, a plate coated with an antigen (e.g., recombinant TfR) is incubated with a sample containing an anti-TfR antibody, and the binding of the antigen to the antigen of interest is determined.

Assays for evaluating uptake of systemically administered antibodies and other biological activities of the antibodies can be performed as disclosed in the Examples or as known for the anti-CNS antigen antibodies of interest.

In one aspect, there is provided an assay for identifying an anti-TfR antibody conjugated to (covalently or non-covalently) an anti-BACE1 antibody with biological activity. Biological activity can include, for example, suppressing BACE1 aspartyl protease activity. There is also provided an antibody with such biological activity in vivo and/or in vitro, for example, as determined by homogeneous time-resolved fluorescence HTRF or microfluidic capillary electrophoresis (MCE) using a synthetic substrate peptide evaluation or in vivo evaluation in a cell line expressing a BACE1 substrate such as APP.

F. Pharmaceutical Preparations

Pharmaceutical formulations of anti-TfR antibodies as described herein are prepared by mixing the antibody of the desired purity with one or more optional pharmaceutical carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of a lyophilized formulation or an aqueous solution. Pharmaceutical carriers, excipients, or stabilizers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexahydroxyquaternium chloride, benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 Residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other sugars including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutical carriers herein also include interstitial drug dispersants such as soluble neutral-active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more other glycosaminoglycanases, such as chondroitinase.

Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO 2006/044908, the latter formulation including a histidine-acetate buffer.

The preparations herein may also comprise more than one active ingredient, the active ingredient being required for the particular indication being treated, preferably having those active ingredients that do not adversely affect the complementary activities of each other. For example, one or more active ingredients for the treatment of neurological conditions, neurodegenerative diseases, cancer, ophthalmopathy, epileptic seizure conditions, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders or CNS inflammation may be desirably provided in addition. Exemplary such drugs are discussed hereinafter herein. The active ingredients are suitably combined in an amount effective for the intended use.

The active ingredient can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, in hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. For example, these techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980). One or more active ingredients can be encapsulated in liposomes coupled to an anti-TfR antibody described herein (e.g., see U.S. Patent Application Publication No. 20020025313).

Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which are in the form of formed articles, such as films or microcapsules. Non-limiting examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Patent number 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT ^™ (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid.

Formulations intended for in vivo administration are generally sterile. Sterility can be readily accomplished, for example, by filtration through sterile filtration membranes.

G. Methods of Treatment and Compositions

Any anti-TfR antibody provided herein can be used in therapeutic methods. In one aspect, an anti-TfR antibody for use as a drug is provided. For example, the present invention provides a method for transporting a therapeutic compound across the blood-brain barrier, the method having a reduced or eliminated effect on the red blood cell population, the method comprising exposing an anti-TfR antibody (e.g., a multispecific antibody that binds both TfR and a brain antigen) coupled to the therapeutic compound to the BBB so that the antibody transports the therapeutic compound coupled thereto across the BBB. In another example, the present invention provides a method for transporting a neurological disease drug across the blood-brain barrier, the method comprising exposing an anti-TfR antibody (e.g., a multispecific antibody that binds both TfR and a brain antigen) of the present invention coupled to a brain disorder drug to the BBB so that the antibody transports the neurological disease drug coupled thereto across the BBB and has a reduced or eliminated effect on the red blood cell population. In one embodiment, the BBB is in a mammal (e.g., a human), e.g., a mammal (e.g., a human) with a neurological disease, including, but not limited to, Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman's syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, traumatic brain injury, etc.

In one embodiment, the neurological disease is selected from the group consisting of: neuropathy, amyloidosis, cancer (e.g., involving the CNS or brain), an eye disease or disorder, a viral or microbial infection, inflammation (e.g., inflammation of the CNS or brain), ischemia, a neurodegenerative disease, a seizure, a behavioral disorder, a lysosomal storage disease, etc. The antibodies of the invention are particularly suitable for treating such neurological diseases due to their ability to transport one or more associated active ingredients/conjugated therapeutic compounds across the BBB and into the CNS/brain where the disorder finds its molecular, cellular or viral/microbial basis.

Neurological disorders are diseases or disorders of the nervous system characterized by inappropriate or uncontrolled neural signaling or absent signaling, and include, but are not limited to, chronic pain (including nociceptive pain), pain caused by injury to body tissue, including cancer-related pain, neuropathic pain (pain caused by abnormalities in the nerves, spinal cord, or brain), and psychogenic pain (related entirely or largely to a psychological disorder), headaches, migraines, neuropathies, and the symptoms and syndromes that often accompany such neurological disorders, such as vertigo or nausea.

For neurological conditions, neurological drugs that can be selected as analgesics include, but are not limited to, narcotic/opioid analgesics (i.e., morphine, fentanyl, hydrocodone, meperidine, methadone, oxymorphone, pentazocine, propoxyphene, tramadol, codeine, and oxycodone), nonsteroidal anti-inflammatory drugs (NSAIDs), and narcotics. NSAIDs (i.e., ibuprofen, naproxen, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, indomethacin, ketorolac, mefenamic acid, meloxicam, nabumetone, oxaprozin) (oxaprozin, piroxicam, sulindac, and tolmetin), corticosteroids (i.e., cortisone, prednisone, prednisolone, dexamethasone, methylprednisolone, and triamcinolone), antimigraine agents (i.e., sumatriptin, almotriptan, ptan, frovatriptan, sumatriptan, rizatriptan, eletriptan, zolmitriptan, dihydroergotamine, eletriptan and ergotamine), acetaminophen, salicylates (i.e., aspirin, choline salicylate, magnesium salicylate), salicylate, diflunisal, and salsalate), anticonvulsants (i.e., carbamazepine, clonazepam, gabapentin, lamotrigine, pregabalin, tiagabine, and topiramate), anesthetics (i.e., isoflurane, trichloroethylene, halothane, sevoflurane, benzocaine, chloroprocaine, cocaine, cyclomethycaine, dimethocaine, propoxycaine, procaine), Cacaine, novocaine, proparacaine, tetracaine, articaine, bupivacaine, carticaine, cinchocaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, piperocaine, prilocaine, ropivacaine, trimecaine, saxitoxin, and tetrodotoxin), and cox-2 inhibitors (i.e., celecoxib, rofecoxib, and valdecoxib). For neuropathic conditions accompanied by vertigo, neurological drugs that can be selected as anti-vertigo agents include, but are not limited to, meclizine, diphenhydramine, promethazine, and diazepam. For neurological conditions accompanied by nausea, neurological drugs that can be selected as anti-nausea agents include, but are not limited to, promethazine, chlorpromazine, prochlorperazine, trimethobenzamide, and metoclopramide.

Amyloidosis is a group of diseases and disorders associated with extracellular proteinaceous deposits in the CNS, including, but 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), Guam Parkinson-Dementia complex, cerebral amyloid angiopathy, Huntington's disease, progressive supranuclear palsy, multiple sclerosis, Creutzfeld-Jakob disease, Parkinson's disease, transmissible spongiform encephalopathy, and encephalopathy. encephalopathy, HIV-associated dementia, amyotrophic lateral sclerosis (ALS), inclusion-body myositis (IBM), and eye diseases involving β-amyloid deposition (ie, macular degeneration, drusen-associated optic neuropathy, and cataracts).

For amyloidosis, neurological drugs may be selected that include, but are not limited to, antibodies or other binding molecules (including, but not limited to, small molecules, peptides, aptamers, or other protein binders) that specifically bind to a target selected from β-secretase, tau, presenilin, amyloid precursor protein or a portion thereof, amyloid β peptide or an oligomer or fibril thereof, death receptor 6 (DR6), receptor for advanced glycation end products (RAGE), parkin, and huntingtin; cholinesterase inhibitors (i.e., galantamine, donepezil, rivastigmine, and tacrine); NMDA receptor antagonists (i.e., memantine), monoamine depleting agents (i.e., tadalafil, dapoxetine, and tadalafil); depletor (i.e., tetrabenazine); ergoloid mesylate; anticholinergic antiparkinsonian drugs (i.e., procyclidine, diphenhydramine, trihexylphenidyl, benztropine, biperiden, and trihexyphenidyl); dopaminergic antiparkinsonian drugs (i.e., entacapone, selegiline, pramipexole, bromocriptine, rotigotine, selegiline, ropinirole, rasagiline, apomorphine, carbidopa, levodopa, pergolide, tolcapone, and amantadine); tetrabenazine; anti-inflammatory drugs (including, but not limited to, nonsteroidal anti-inflammatory drugs (i.e., indomethicin and other compounds listed above); hormones (i.e., estrogens, progesterone, and leuprolide); vitamins (i.e., folic acid and niacinamide); dimebolin; homotaurine (i.e., 3-aminopropanesulfonic acid; 3APS); serotonin receptor activity modulators (i.e., xaliproden); interferons, and glucocorticoids.

Cancers of the CNS are characterized by abnormal proliferation of one or more CNS cells (i.e., nerve cells) and include, but are not limited to, gliomas, glioblastoma multiforme, meningiomas, astrocytomas, acoustic neuromas, chondromas, oligodendrogliomas, medulloblastomas, gangliogliomas, schwannomas, neurofibromas, neuroblastomas, and epidural, intramedullary, or intradural tumors.

For cancer, a neurological drug that is a chemotherapeutic agent may be selected. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelam ine), triethylenephosphoramide, triethiylenethiophosphor-amide, and trimethylomelamine; polyacetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol); beta-lapachone; lapachol; colchicines; betulinic acid acid; camptothecin (including the synthetic analogs topotecan, CPT-11 (irinotecan), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllinic acid acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, and chlorambucil. hydrochloride), melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1I and 1,1-diyne); Calicheamicin ωI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); enediyne anthracyclines, including enediyne anthracycline A; esperamicin; and neocarzinostatin chromophores and related pigment proteins enediyne antibiotic chromophores), aclacinomycins, actinomycins ), anthramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin), 2-pyrrolino-doxorubicin, and deoxydoxorubicin. ubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil ( 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone and dromostanolone propionate propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycoside; 5-aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; polysaccharide complexes (JHS Natural Products (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verracurin A, roridin A, and roridin B); A) and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, such as paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, an albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and docetaxel (Poulenc Oncology, Princeton, N.J.). Rorer, Antony, France); chloranbucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide mide; mitoxantrone; vincristine; oxaliplatin; leucovorin; vinorelbine; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitors (RFS) 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; a pharmaceutically acceptable salt, acid or derivative of any of the foregoing; and combinations of two or more of the foregoing, such as CHOP, which is an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, which is an abbreviation for the treatment regimen of oxaliplatin combined with 5-FU and leucovorin (ELOXATIN™).

Also included in this definition of chemotherapeutic agents are antihormonal agents, which act to modulate, reduce, block or inhibit the effects of hormones that promote cancer growth, and are usually in the form of systemic or whole body treatments. They can themselves be hormones. Examples include antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene); antiprogestins; estrogen receptor downregulators (ERDs); agents that act to suppress or shut down the ovaries, for example, luteinizing hormone-releasing hormone (LHRH) agonists, such as leuprolide acetate, goserelin acetate, and leuprolide acetate. and tripterelin acetate, buserelin acetate, and tripterelin; other antiandrogens, such as flutamide, nilutamide, and bicalutamide; and aromatase inhibitors, which inhibit the aromatase enzyme, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, and anastrozole. In addition, this definition of chemotherapeutic agents includes bisphosphonates, such as clodronate (e.g., clodronate or etidronate), NE-58095, zoledronic acid/zoledronate, alendronate, pamidronate, tiludronate, or risedronate; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as vaccines and gene therapy vaccines, for example, vaccines, vaccines, and vaccines; topoisomerase 1 inhibitors; rmRH; lapatinib; ditosylate) (a small molecule inhibitor of ErbB-2 and EGFR dual tyrosine kinases, also known as GW572016); and pharmaceutically acceptable salts, acids or derivatives of any one of the above.

Another group of compounds that can be selected for use in neurological drugs for cancer treatment or prevention are anticancer immunoglobulins (including but not limited to trastuzumab, pertuzumab, bevacizumab, alemtuxumab, cetuximab, gemtuzumab ozogamicin, ibritumomab tiuxetan, panitumumab and rituximab). In some cases, antibodies bound to toxic markers or conjugates can be used to target and kill target cells (i.e., cancer cells), including but not limited to tositumomab with ¹³¹ I radiolabel, or trastuzumab emtansine.

An ocular disease or disorder is a disease or disorder of the eye, which for purposes herein is considered to be an organ of the CNS subject to the BBB. Ocular diseases or disorders include, but are not limited to, disorders of the sclera, cornea, iris, and ciliary body (i.e., scleritis, keratitis, corneal ulcer, corneal abrasion, snow blindness, arc eye, Thygeson's superficial punctate keratopathy, corneal neovascularization, Fuchs' dystrophy, keratoconus, keratoconjunctivitis sicca, iritis, and uveitis), disorders of the lens (i.e., cataract), disorders of the choroid and retina (i.e., retinal detachment), and ocular diseases or disorders of the iris (i.e., cataract). detachment, retinoschisis, hypertensive retinopathy, diabetic retinopathy, retinopathy, retinopathy of prematurity, age-related macular degeneration, macular degeneration (wet or dry), epiretinal membrane, retinitis pigmentosa and macular edema), glaucoma, floaters, disorders of the optic nerve and visual pathway (i.e., Leber's hereditary optic neuropathy and optic disc drusen), discdrusen), disorders of ocular muscle/binocular movement accommodation/refraction (i.e., strabismus, ophthalmoparesis, progressive external ophthalmoplegia, esotropia, exotropia, hyperopia, myopia, astigmatism, refractive error, presbyopia, and ophthalmoplegia), visual impairment and blindness (i.e., amblyopia, Lever's congenital amaurosis, scotoma, color blindness, achromatopsia, nyctalopia, blindness, river blindness, and micro-opthalmia/coloboma), red eye, Argyll Robertson pupil, keratomycosis, dry eye, and aniridia.

For ocular diseases or conditions, neurological drugs that may be selected are: anti-neovascular ophthalmic agents (i.e., bevacizumab, ranibizumab, and pegaptanib), ophthalmic glaucoma drugs (i.e., carbachol, epinephrine, demecarium bromide, apraclonidine, brimonidine, brinzolamide, levobunolol), and sedatives. vobunolol, timolol, betaxolol, dorzolamide, bimatoprost, carteolol, metipranolol, dipivefrin, travoprost, and latanoprost), carbonic anhydrase inhibitors (ie, methazolamide and acetazolamide), ophthalmic antihistamines ( i.e., naphazoline, phenylephrine, and tetrahydrozoline), ophthalmic lubricants, ophthalmic steroids (i.e., fluorometholone, prednisolone, loteprednol, dexamethasone, difluprednate, rimexolone, fluocinolone, medrysone, rysone and triamcinolone), ophthalmic anesthetics (i.e., lidocaine, proparacaine, and tetracaine), ophthalmic anti-infectives (i.e., levofloxacin, gatifloxacin, ciprofloxacin, moxifloxacin, chloramphenicol, bacitracin/polymyxin B), b), sulfacetamide, tobramycin, azithromycin, besifloxacin, norfloxacin, sulfisoxazole, gentamicin, idoxuridine, erythromycin, natamycin, gramicidin, neomycin, ofloxacin, trifluridine, ganciclovir, vidarabine), ophthalmic anti-inflammatory agents (i.e., nepafenac, ketorolac, flurbiprofen, suprofen, cyclosporine, triamcinolone, diclofenac, and bromfenac), and ophthalmic antihistamines or decongestants (i.e., ketotifen, olopatadine, epinastine, naphazoline, cromolyn, tetrahydrozoline, pemiirolast, bepotastine, naphazoline, phenylephrine, nedocromil, lodoxamide, phenylephrine, emedastine, and azelastine).

Viral or microbial infections of the CNS include, but are not limited to, infections caused by viruses (i.e., influenza, HIV, poliovirus, rubella), bacteria (i.e., Neisseria sp., Streptococcus sp., Pseudomonas sp., Proteus sp., E. coli, S. aureus, Pneumococcus sp., Meningococcus sp., Haemophilus sp., and Mycobacterium tuberculosis), and other microorganisms, such as fungi (i.e., yeast, Cryptococcus neoformans), parasites (i.e., toxoplasma gondii), and nematodes. gondii) or amoebas, leading to CNS pathophysiology including, but not limited to, meningitis, encephalitis, myelitis, vasculitis, and abscesses, which may be acute or chronic.

For viral or microbial diseases, neurological drugs may be selected that include, but are not limited to, antiviral compounds (including, but not limited to, adamantane antivirals (i.e., rimantadine and amantadine), antiviral interferons (i.e., peginterferon alfa-2b), chemokine receptor antagonists (i.e., maraviroc), integrase strand transfer inhibitors (i.e., raltegravir), neuraminidase inhibitors (i.e., oseltamivir and zanamivir), non-nucleoside reverse transcriptase inhibitors (i.e., efavirenz, etravirine, delavirdine), and sirolimus. virdine and nevirapine), nucleoside reverse transcriptase inhibitors (dodanosine plus tenofovir, abacavir, lamivudine, zidovudine, stavudine, entecavir, emtricitabine, adefovir, zalcitabine, telbivudine, telbivudine and didanosine), protease inhibitors (i.e., darunavir, atazanavir, fosamprenavir, tipranavir, ritonavir, nelfinavir, amprenavir, indinavir, and saquinavir), ir), purine nucleosides (i.e., valacyclovir, famciclovir, acyclovir, ribavirin, ganciclovir, valganciclovir, and cidofovir), and miscellaneous antivirals (i.e., enfuvirtide, foscarnet, palivizumab, vizumab and fomivirsen), antibiotics (including but not limited to aminopenicillins (i.e., amoxicillin, ampicillin, oxacillin, nafcillin, cloxacillin, dicloxacillin, flucoxacillin, temocillin, azlocillin, carbenicillin, ticarcillin, mezlocillin, piperacillin, and bacampicillin), cephalosporins (i.e., cefazolin, cephalexin, cephalothin, cefmandole), andole, ceftriaxone, cefotaxime, cefpodoxime, ceftazidime, cefadroxil, cephradine, loracarbef, cefotetan, cefuroxime, cefprozil, cefaclor or and cefoxitin), carbapenem/penem (i.e., imipenem, meropenem, ertapenem, faropenem, and doripenem), monobactam (i.e., aztreonam, tigemonam, norcardicin A, and tabtoxinine-β-lactam), β-lactamase inhibitors in combination with another β-lactam antibiotic (i.e., clavulanic acid), acid, tazobactam, and sulbactam), aminoglycosides (i.e., amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, and paromomycin), ansamycins (i.e., geldanamycin and herbimycin), carbacephems (i.e., loracarbef), glycopeptides (i.e., teicoplanin and vancomycin), macrolides (i.e., azithromycin, clarithromycin), arithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, and spectinomycin), monobactams (i.e., aztreonam), quinolone (i.e., ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin), acin, grepafloxacin, sparfloxacin, and temafloxacin), sulfonamides (i.e., mafenide, sulfonamidochrysoidine, sulfacetamide, sulfadiazine, sulfamethizole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim, and sulfamethoxazole), tetracyclines (i.e., tetracycline, demeclocycline, meclocycline, doxycycline, minocycline, and oxytetracycline), antineoplastic or cytotoxic antibiotics (i.e., doxorubicin, mitoxantrone, bleomycin, daunorubicin, dactinomycin, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin, and valrubicin), and miscellaneous antibacterial compounds (i.e., bacitracin, colistin, and polymyxin B). B)), antifungals (i.e., metronidazole, nitazoxanide, tinidazole, chloroquine, iodoquinol, and paromomycin), and antiparasitics (including, but not limited to, quinine, chloroquine, amodiaquine, pyrimethamine, sulphadoxine, proguanil, mefloquine, atovaquone, primaquine, artemesinin, halofantrine, doxycycline, clindamycin, mebendazole, pyrantel pamoate, pamoate, thiabendazole, diethylcarbamazine, ivermectin, rifampin, amphotericin B, melarsoprol, efornithine, and albendazole).

Inflammation of the CNS includes, but is not limited to, inflammation resulting from injury to the CNS, which may be physical (i.e., due to accident, surgery, brain trauma, spinal cord injury, concussion) or injury resulting from or associated with one or more other diseases or conditions of the CNS (i.e., abscess, cancer, viral or microbial infection).

For CNS inflammation, one may choose a neurological drug that treats the inflammation itself (ie, nonsteroidal anti-inflammatory agents such as ibuprofen or naproxen) or a neurological drug that treats the underlying cause of the inflammation (ie, antiviral or anticancer agents).

As used herein, ischemia of the CNS refers to a group of disorders that are related to abnormal blood flow or vascular behavior in the brain, or their causes, and include, but are not limited to, focal brain ischemia, global brain ischemia, stroke (i.e., subarachnoid hemorrhage and intracerebral hemorrhage), and aneurysm.

For ischemia, neurological drugs that may be selected include, but are not limited to, thrombolytics (i.e., urokinase, alteplase, reteplase, and tenecteplase), platelet aggregation inhibitors (i.e., aspirin, cilostazol, clopidogrel, prasugrel, and dipyridamole), statins (i.e., lovastatin, pravastatin, fluvastatin, rosuvastatin, atorvastatin, simvastatin, cerivastatin, and pitavastatin), and compounds that increase blood flow or vascular elasticity, including, for example, blood pressure drugs.

Neurodegenerative diseases are a group of diseases and disorders associated with the loss of function or death of nerve cells in the CNS and include, but are not limited to, adrenoleukodystrophy, Alexander disease, Alper's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, Batten disease, Cockayne syndrome, corticobasal degeneration, degeneration caused by or associated with amyloidosis, Friedreich's ataxia, frontotemporal lobar degeneration, Kennedy's disease, multiple system atrophy, multiple sclerosis, primary lateral sclerosis, progressive supranuclear palsy, spinal muscular atrophy, transverse myelitis, Refsum's disease, and spinocerebellar ataxia.

For neurodegenerative diseases, neurological drugs that are growth hormones or neurotrophic factors can be selected; examples include, but are not limited to, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-4/5, fibroblast growth factor (FGF)-2 and other FGFs, neurotrophin (NT)-3, erythropoietin (EPO), hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor (TGF)-α, TGF-β, vascular endothelial growth factor (VEGF), interleukin-1 receptor antagonist (IL-1ra), ciliary neurotrophic factor (CNTF), glial-derived GDNF, neurturin, platelet-derived growth factor (PDGF), heregulin, neuregulin, artemin, persephin, interleukins, glial cell line-derived neurotrophic factor (GFR), granulocyte colony-stimulating factor (CSF), granulocyte-macrophage-CSF, netrin, cardiotrophin-1, hedgehogs, leukemia inhibitory factor (LIF), midkine, pleiotrophin, bone morphogenetic protein (BMP), netrin, saponin, semaphorin, and stem cell factor (SCF).

Seizure diseases and disorders of the CNS involve inappropriate and/or abnormal electrical conduction in the CNS and include, but are not limited to, epilepsy (i.e., absence seizures, atonic seizures, benign motor epilepsy, childhood absence, clonic seizures, complex partial seizures, frontal lobe epilepsy, febrile seizures, infantile spasms, juvenile myoclonic epilepsy, juvenile absence epilepsy, Lennox-Gastaut syndrome, Landau-Kleffner syndrome, Syndrome, Dravet's syndrome, Otahara syndrome, West syndrome, myoclonic seizures, mitochondrial disorders, progressive myoclonic epilepsies, psychogenic seizures, reflex epilepsy, Rasmussen's syndrome, simple partial seizures, secondarily generalized seizures, temporal lobe epilepsy, toniclonic seizures, tonic seizures, psychomotor seizures, limbic epilepsy, partial-onset seizures seizures, generalized-onset seizures, status epilepticus, abdominal epilepsy, akinetic seizures, autonomic seizures, massive bilateral myoclonus, catamenial epilepsy, drop seizures, emotional seizures, focal seizures, gelastic seizures, Jacksonian March, Lafora Disease, motor seizures, multifocal seizures, nocturnal seizures, photosensitive seizures, pseudoseizures seizures, sensory seizures, subtle seizures, sylvan seizures, withdrawal seizures, and visual reflex seizures.

For seizure disorders, neurological drugs that are anticonvulsants or antiepileptic drugs may be selected, including but not limited to barbiturate anticonvulsants (i.e., primidone, metharbital, mephobarbital, allobarbital, amobarbital, aprobarbital, alphenal, barbital, brallobarbital, and phenobarbital), benzodiazepine anticonvulsants (i.e., diazepam, clonazepam, and lorazepam), carbamate anticonvulsants (i.e., felbamate), carbonic anhydrase inhibitors (i.e., phenobarbital), and benzodiazepine anticonvulsants. anhydrase inhibitor anticonvulsants (i.e., acetazolamide, topiramate, and zonisamide), dibenzazepine anticonvulsants (i.e., rufinamide, carbamazepine, and oxcarbazepine), fatty acid derivative anticonvulsants (i.e., divalproex and valproic acid), acid), GABA analogs (i.e., pregabalin, gabapentin, and vigabatrin), GABA reuptake inhibitors (i.e., tiagabine), GABA transaminase inhibitors (i.e., vigabatrin), hydantoin anticonvulsants (i.e., phenytoin, ethotoin, fosphenytoin, and mephenytoin), miscellaneous anticonvulsants (i.e., lacosamide and magnesium sulfate) , progestins (i.e., progesterone), oxazolidinedione anticonvulsants (i.e., paramethadione and trimethadione), pyrrolidine anticonvulsants (i.e., levetiracetam), succinimide anticonvulsants (i.e., ethosuximide and methsuximide), triazine anticonvulsants (i.e., lamotrigine), and urea anticonvulsants (i.e., phenacemide and pheneturide).

Behavioral disorders are disorders of the CNS characterized by abnormal behavior in the afflicted subject and include, but are not limited to, sleep disorders (i.e., insomnia, parasomnias, night terrors, circadian rhythm sleep disorders, and narcolepsy), mood disorders (i.e., depression, suicidal depression, anxiety, chronic affective disorders, 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, disorder), eating disorders (i.e., anorexia or bulimia), major mental illnesses (psychoses), developmental behavioral disorders (i.e., autism, Rett’s syndrome, Aspberger’s syndrome), personality disorders, and psychotic disorders (i.e., schizophrenia, delusional disorder, etc.).

For behavioral disorders, the neurological drug can be selected from behavior-modifying compounds including, but not limited to, atypical antipsychotics (i.e., risperidone, olanzapine, apripiprazole, quetiapine, paliperidone, asenapine, clozapine, iloperidone, and ziprasidone), phenothiazine antipsychotics (i.e., prochlorperazine, prochlorperazine, chlorpromazine, fluphenazine, perphenazine, trifluoperazine, thioridazine, and mesoridazine), thioxanthines (i.e., thiothixene), miscellaneous antipsychotics (i.e., pimozide, lithium, molindone, haloperidol and loxapine), selective serotonin reuptake inhibitors (ie, citalopram, escitalopram, paroxetine, fluoxetine, and sertraline), serotonin-norepinephrine reuptake inhibitors (ie, duloxetine, venlafaxine, desvenlafaxine), desvenlafaxine), tricyclic antidepressants (i.e., doxepin, clomipramine, amoxapine, nortriptyline, amitriptyline, trimipramine, imipramine, protriptyline, and desipramine), tetracyclic antidepressants (i.e., mirtazapine, zapine and maprotiline), phenylpiperazine antidepressants (i.e., trazodone and nefazodone), monoamine oxidase inhibitors (i.e., isocarboxazid, phenelzine, selegiline, and tranylcypromine), benzodiazepines (i.e., alprazolam, estazolam, flurazepam), ptam, clonazepam, lorazepam, and diazepam), norepinephrine-dopamine reuptake inhibitors (i.e., bupropion), CNS stimulants (i.e., phentermine, diethylpropion, methamphetamine, dextroamphetamine, amphetamine, methylphenidate), thylphenidate, dexmethylphenidate, lisdexamfetamine, modafinil, pemoline, phendimetrazine, benzphetamine, phendimetrazine, armodafinil, diethylpropion, caffeine, atomoxetine, doxapram, and mazindol), anxiolytics/sedatives/hypnotics (including, but not limited to, barbiturates (i.e., secobarbital, phenobarbital, and mephobarbital), benzodiazepines (as described above), and miscellaneous anxiolytics/sedatives/hypnotics (i.e., diphenhydramine, sodium oxybate, oxybate, zaleplon, hydroxyzine, chloral hydrate, aolpidem, buspirone, doxepin, eszopiclone, ramelteon, meprobamate, and ethclorvynol), secretins (see, e.g., Ratliff-Schaub et al., Autism 9:256-265 (2005)), opioid peptides (see, e.g., Cowen et al., J. Neurochem. 89:273-285 (2004)), and neuropeptides (see, e.g., Hethwa et al., Am. J. Physiol. 289:E301-305 (2005)).

Lysosomal storage disorders are metabolic disorders that, in some cases, are associated with the CNS or have CNS-specific symptoms; such disorders include, but are not limited to, Tay-Sachs disease, Gaucher's disease, Fabry disease, mucopolysaccharidosis (types I, II, III, IV, V, VI, and VII), glycogen storage disease, GM1-gangliosidosis, metachromatic leukodystrophy, Farber's disease, Canavan's leukodystrophy, and neuronal ceroid lipofuscinoses types 1 and 2, Niemann-Pick disease, Pompe disease, and disease, and Krabbe’s disease.

For lysosomal storage diseases, neurological drugs may be selected that either themselves or otherwise mimic the activity of the enzyme that is impaired in the disease. Exemplary recombinant enzymes for treating lysosomal storage diseases include, but are not limited to, those described, for example, in U.S. Patent Application Publication No. 2005/0142141 (i.e., α-L-iduronidase, iduronate-2-sulfatase, N-sulfatase, α-N-acetylglucosaminidase, N-acetyl-galactosamine-6-sulfatase, β-galactosidase, arylsulfatase B, β-glucuronidase, acid α-glucosidase, glucocerebrosidase, α-galactosidase A, hexosaminidase A, acid sphingomyelinase, β-galactocerebrosidase, β-galactosidase, arylsulfatase A, acid amidase, aspartoacylase, palmitoyl-protein thioesterase 1, and tripeptidyl aminopeptidase 1).

In another embodiment, diseases associated with or caused by inappropriate overproduction of red blood cells, or diseases in which overproduction of red blood cells is a function of the disease, can be prevented or treated by the reticulocyte depletion effect recognized herein of anti-TfR antibodies that retain at least partial effector function. For example, in congenital or neoplastic erythrocytosis, elevated red blood cell counts due to, for example, excessive proliferation of reticulocytes lead to blood thickening and accompanying physiological symptoms (d'Onofrio et al., Clin. Lab. Haematol. (1996) Suppl. 1: 29-34). Administration of the anti-TfR antibodies of the present invention in which at least partial effector function of the antibody is retained will allow selective removal of immature reticulocyte populations without affecting normal transferrin transport to the CNS. As is well known in the art, the administration of such antibodies can be adjusted so that acute clinical symptoms can be minimized (i.e., at very low doses or at wide intervals).

In one aspect, the antibodies of the invention are used to detect neurological diseases and/or assess the severity or duration of a disease or condition before the onset of symptoms. In one aspect, the antibodies allow for the detection and/or imaging of neurological diseases, including imaging by radiography, tomography, or magnetic resonance imaging (MRI).

In one aspect, a low-affinity anti-TfR antibody of the present invention is provided for use as a drug. In another aspect, a low-affinity anti-TfR antibody for treating a neurological disease or disorder (e.g., Alzheimer's disease) without consuming red blood cells (i.e., reticulocytes) is provided. In certain embodiments, a low-affinity anti-TfR antibody for modification in a method of treating a subject as described herein is provided. In certain embodiments, the present invention provides a low-affinity anti-TfR antibody modified to improve its safety, which is used in a method for treating an individual with a neurological disease or disorder, the method comprising administering an effective amount of an anti-TfR antibody (optionally coupled to a neurological disease drug) to the individual. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual. In another embodiment, the present invention provides an anti-TfR antibody modified to improve its safety, which is used to reduce or inhibit amyloid plaque formation in a patient at risk of a neurological disease or disorder (e.g., Alzheimer's disease) or suffering from the disease or disorder. An "individual" according to any of the above embodiments is optionally a human. In certain aspects, the anti-TfR antibodies of the invention used in the methods of the invention increase the uptake of a neurological disease drug to which it is conjugated.

In another aspect, the present invention provides the use of a low-affinity anti-TfR antibody of the present invention in the production or preparation of a medicament. In one embodiment, the medicament is for treating a neurological disease or disorder. In another embodiment, the medicament is for use in a method for treating a neurological disease or disorder, the method comprising administering an effective amount of the medicament to an individual suffering from the neurological disease or disorder. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual.

In another aspect, the present invention provides a method for treating Alzheimer's disease. In one embodiment, the method comprises administering to an individual suffering from Alzheimer's disease an effective amount of a multispecific antibody of the present invention that binds both BACE1 and TfR or both Aβ and TfR. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. The "individual" according to any of the above embodiments may be a human.

In therapy, the anti-TfR antibodies of the present invention can be used alone or in combination with other agents. For example, the anti-TfR antibodies of the present invention can be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is a therapeutic agent that is effective for treating a neurological disease that is the same as or different from the neurological disease being treated with the anti-TfR antibody. Exemplary additional therapeutic agents include, but are not limited to, the various neurological drugs described above, cholinesterase inhibitors (such as donepezil, galantamine, rivastigmine, and tacrine), NMDA receptor antagonists (such as memantine), amyloid beta peptide aggregation inhibitors, antioxidants, γ-secretase modulators, nerve growth factor (NGF) mimetics or NGF gene therapy, PPARγ agonists, HMS-CoA reductase inhibitors (statins), ampakines, calcium channel blockers, GABA receptor antagonists, glycogen synthase kinase inhibitors, intravenous immunoglobulin, muscarinic receptor agonists, nicotine receptor modulators, active or passive amyloid beta 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 alleviate one or more side effects of a neurological drug.

As exemplified herein, certain anti-TfR antibodies may have side effects that adversely affect the reticulocyte population in subjects treated with the anti-TfR antibodies. Thus, in certain embodiments, at least one additional therapeutic agent selected for its ability to mitigate such adverse side effects on the reticulocyte population is co-administered with the anti-TfR antibodies of the present invention. Examples of such therapeutic agents include, but are not limited to, agents that increase the red blood cell (i.e., reticulocyte) population, agents that support the growth and development of red blood cells (i.e., reticulocytes), and agents that protect the red blood cell population from the effects of anti-TfR antibodies; such 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 (i.e., reticulocytes) by, for example, transfusion with similar cells that may be from another individual with a similar blood type or that may have been previously extracted from a subject to whom the anti-TfR antibody is administered. One of ordinary skill in the art will understand that, in some instances, it is preferred to administer to a subject an agent intended to protect existing red blood cells (i.e., reticulocytes) prior to or concurrently with anti-TfR antibody treatment, while it is preferred to administer to a subject an agent intended to support or initiate regrowth/development of red blood cells or blood cell populations (i.e., reticulocytes or reticulocyte populations) concurrently with or subsequent to anti-TfR antibody treatment so that such blood cells can be replenished following anti-TfR antibody treatment.

In certain other such embodiments, at least one additional therapeutic agent is selected for its ability to inhibit or prevent activation of the complement pathway upon administration of the anti-TfR antibody. Examples of such therapeutic agents include, but are not limited to, agents that interfere with the ability of the anti-TfR antibody to bind to or activate the complement pathway and agents that inhibit one or more molecular interactions within the complement pathway and are generally described in Mollnes and Kirschfink (2006) Molec. Immunol. 43: 107-121, the contents of which are expressly incorporated herein by reference.

Such combination therapies described above and herein include combined administration (wherein two or more therapeutic agents are contained in the same or separate formulations), and separate administration, wherein administration of the antibody of the invention can occur before, simultaneously with, and/or after administration of the additional therapeutic agent and/or adjuvant. In one embodiment, administration of the anti-TfR antibody and administration of the additional therapeutic agent occur within about one month, or within about one week, two weeks, or three weeks, or within about one day, two days, three days, four days, five days, or six days of each other. The antibodies of the invention can also be used in combination with other interventional therapies, such as, but not limited to, radiation therapy, behavioral therapy, or other therapies known in the art and suitable for the neurological disease to be treated or prevented.

The anti-TfR antibodies of the present invention (and any additional therapeutic agents) can be administered by any suitable method, including parenteral administration, intrapulmonary administration and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Depending on whether the medication is short-term or long-term, the medication can be administered by any suitable route, for example, by injection, such as intravenous or subcutaneous injection. Various dosing regimens are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

The antibodies of the present invention are formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered in this regard include specific conditions to be treated, specific mammals to be treated, the clinical status of individual patients, the cause of the condition, the site of the delivery agent, the method of administration, the administration schedule, and other factors known to medical practitioners. The antibodies do not need to be formulated, but are optionally formulated together with one or more medicaments currently used to prevent or treat the condition in question or to prevent, alleviate, or improve one or more side effects of the antibody administration. The effective amount of the other medicaments depends on the amount of the antibody present in the preparation, the type of condition or treatment, and other factors discussed above. These are generally in the same dose, and use a route of administration as described herein, or with about 1-99% of the dosage as described herein, or with experience/clinically determined to be suitable arbitrary dose and any approach.

For the prevention or treatment of disease, the appropriate dosage of the antibody of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and progression of the disease, whether the antibody is administered for preventive purposes or for therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient in a single treatment or over a series of treatments. Depending on the type and severity of the disease, an antibody of about 1 μg/kg-15 mg/kg (e.g., 0.1 mg/kg-10 mg/kg) can be an initial candidate dose for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dose can be in the range of about 1 μg/kg-100 mg/kg or more, depending on the factors mentioned above. For repeated administration for several days or longer, depending on the condition, treatment will generally be continued until the desired disease symptom suppression occurs. An exemplary dosage of the antibody should be in the range of about 0.05 mg/kg-about 40 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg or 40 mg/kg (or any combination thereof) can be administered to the patient. Such doses can be administered at intervals, for example weekly or every three weeks (e.g., so that the patient receives about 2 to about 20 or, for example, about 6 doses of the antibody). An initial higher loading dose can be administered, followed by one or more lower doses. However, other treatment regimens may be useful. It should be understood that one way to reduce the effect on the reticulocyte population by administering an anti-TfR antibody is to change the amount or timing of the medication so that there is an overall lower amount of circulating antibody in the bloodstream to interact with the reticulocytes. In a non-limiting example, a lower dose of an anti-TfR antibody can be administered at a higher frequency than the frequency of a higher dose. The dosage used can be balanced between the amount of antibody that needs to be delivered to the CNS (itself related to the affinity of the CNS antigen-specific portion of the antibody), the affinity of the antibody for TfR, and whether one or more compounds that protect red blood cells (i.e., reticulocytes), promote growth and development, or inhibit the complement pathway are co-administered or administered continuously with the antibody. As described herein and as known in the art, the progress of this therapy can be easily monitored by conventional techniques and assays.

It will be understood that any of the above formulations or treatment methods can be performed using the immunoconjugates of the invention in place of or in addition to anti-TfR antibodies.

H. Products

In another aspect of the present invention, an article of manufacture is provided, comprising materials useful for treating, preventing, and/or diagnosing the aforementioned conditions. The article of manufacture comprises a container and a label or package insert on or with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be made of various materials, such as glass or plastic. The container contains a composition, either alone or in combination with another composition that is effective for treating, preventing, and/or diagnosing the condition, and can have a sterile access port (for example, the container can be an IV bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody of the present invention. The label or package insert indicates that the composition is for treating a selected condition. In addition, the article of manufacture may comprise (a) a first container containing a composition comprising an antibody of the present invention; and (b) a second container containing a composition comprising another cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment of the present invention may also include a package insert indicating that the composition can be used to treat a specific condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also include other materials as may be desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

It will be understood that any of the above articles of manufacture may include an immunoconjugate of the invention in place of or in addition to an anti-TfR antibody.

Example

Example 1: Generation, Characterization, and Humanization of Human/Cynomolgus Monkey Cross-Reactive Anti-TFR Antibodies

Initially, a naive antibody phage panning process was performed to identify antibodies that cross-reacted with both human TfR and TfR from cynomolgus monkeys ("cyno") and did not compete with Tf for binding to TfR (Lee et al. JMB (2004) 1073-1093). No such cross-reactive, non-Tf-competing clones were identified from this phage panning process. However, two antibodies were identified that were useful for characterizing subsequently generated hybridoma clones.

Species cross-reactive antibodies (Tf-competing antibodies) that compete with Tf for binding to human or cynomolgus monkey TfR were identified. Another clone's epitope specific for human TfR was mapped to the apical domain of huTfR using a mouse/human chimeric TfR receptor (Figure 1). When the mouse TfR sequence in the apical domain was replaced with huTfR, the apical domain binding clone lost its binding to huTfR.

Then, an immune-based approach was performed to generate cross-reactive anti-human/cynomolgus monkey TfR antibodies. Human TfR extracellular domain ("ecd") containing N-terminal His and human hemochromatin ("HFE") were expressed and purified as described in (Bennet et al., Nature (2000) 403, 46-53). A similar cynomolgus monkey TfR ecd construct was also prepared. Cynomolgus monkey TfR was expressed and purified in a similar manner. Human and cynomolgus monkey cross-reactive TfR antibodies were generated by immunizing the footpads of 5 Balb/C mice with 6 doses (twice a week) containing 2 μg of each of cynomolgus monkey TfR and huTfR ecd. The sera of all mice were FACS positive, and all mice were pooled. 1632 hybridomas were screened, of which 111 were ELISA positive for binding to human and cynomolgus monkey TfR.

In the presence of 1 μM human holo-Tf, the ELISA-positive hybridoma obtained by FACS screening was screened for binding to 293 cells transiently expressing human or cynomolgus monkey TfR. In brief, 293 cells transfected with full-length human or cynomolgus monkey TfR were subjected to FACS analysis using lipofectamin2000 plus (Invitrogen) 48-72h before FACS analysis. Untransfected (control) and transfected 293 cells were washed twice with FACS buffer (PBS containing 1% BSA), and 50 μl hybridoma supernatants (normalized to 10 μg/ml) were added to 293 cells in the presence of 1 μM human holo-Tf and incubated on ice for 30 minutes. The cells were washed twice with FACS buffer, 50 μl PE-goat-anti-mouse Fcγ (Jackson ImmunoResearch) were added to the cells, and they were incubated on ice for 30 minutes. Cells were washed with FACS buffer and resuspended in 100 μl FACS buffer for analysis.

Fourteen clones were positive for binding to both human and cynomolgus monkey TfR (Figures 2A and 2B). These clones were further subcloned and evaluated for binding to both human and cynomolgus monkey TfR by ELISA, and the epitope was mapped to huTfR using the apical binding phage clones identified above. Briefly, apical domain phage competition ELISA was performed in maxisorp plates coated overnight at 4°C with 2 μg/ml purified human or cynomolgus monkey TfR (in PBS). The plates were washed with PBS/0.05% Tween20 and blocked with Superblock with casein (Thermo Scientific, Hudson, NH). 30 μl aliquots of hybridoma supernatant (standardized to 10 μg/ml) were added to each well for 45 minutes. 30 μl of apical domain-binding phage at OD 0.05 were then added for 15 minutes. The plates were washed with PBS/0.05% Tween 20 and a 1:1000 dilution of HRP-mouse-anti-M13 (GE healthcare) was added to the plates and incubated for 1 hour at room temperature. The plates were washed with PBS/0.05% Tween 20 and bound phage was detected using TMB substrate (BioFX Laboratories, Owings Mills). Nine of the fourteen clones were found to block the binding of the apical binding antibody displayed on the phage (see Figure 2C).

Antibody affinity was measured using surface plasmon resonance ("SPR") (Biacore ^™ , GE Healthcare). Anti-His antibodies (Qiagen) were coupled to four different flow cells of a BIACORE ^™ CM5 sensor chip (Biacore, Inc., Piscataway, NJ) at 6000-8000 RU. Immobilization was achieved by random coupling via amino groups using the process provided by the supplier. 10X HBS-P (Biacore, Inc., Piscataway, NJ) was diluted in water and used as a diluent and running buffer. Purified human or cynomolgus monkey TfR was captured, followed by a 3-fold dilution series of IgG or Fab, which was injected at a flow rate of 30 ml/min using a single cycle kinetic method. Affinity constants were determined using a simple 1:1 Langmuir binding model or a steady-state model when _kon or _koff exceeded the detection limit. The equilibrium dissociation constant ( _KD ) was calculated as the ratio of the association rate constant (kon) to _the dissociation rate constant ( _koff ). The results are shown in Figure 2C.

Each hybridoma was cloned. Total RNA was isolated from the hybridoma using the RNeasy mini kit (Qiagen). cDNA was produced using the SMART 5'RACE cDNA amplification kit (Clontech) according to the instructions of the supplier. The variable regions of each antibody were amplified using the UPM (5'oligo) provided in the kit and the 3'oligo annealed to the constant region. The complete PCR product was then cloned into the pCR4Blunt-TOPO vector (Invitrogen) for sequencing. After sequence analysis, hybridomas can be subdivided into 4 groups (Fig. 3A-3D). The clones competing with the top binding antibody fell into 3 related categories (Fig. 3A-C). 4 non-top clones (Fig. 3D) were composed of 2 related clones and 2 other unique sequences. The light chain and heavy chain CDRs of each clone are provided in Table 3.

Table 3: Light and heavy chain CDRs of cross-reactive anti-cynomolgus monkey/human TfR antibodies

In this article, representative clones of each species (15G11, 7A4, 16F6 and 7G7) were used as examples for humanization and further characterization. Humanization was achieved using HVR transplantation and fine-tuning positions (vernier positions) including those shown below and in Figures 4A-4E. 15G11 was humanized by transplanting HVRs to the IGKV1-NL1*01 and IGHV1-3*01 human variable domains. A combination of different mouse fine-tuning positions was included in the humanized variant, as shown in Figure 4E. The humanized 15G11 variant 15G11.v5 includes selected fine-tuning positions in VL (positions 43 and 48) and VH (positions 48, 67, 69, 71 and 73), as shown in Figure 4A. In addition, the N-terminus of VH was changed from Q to E. To humanize 7A4, HVR grafting was performed using the 7A4 heavy chain and 8A2 light chain HVRs (7A4 and 8A2 are related clones, Figure 3A). The HVRs were grafted into the IGKV4-1*01 and IGHV1-2*02 human variable domains. A combination of different mouse fine-tuning positions was included in the humanized variants, as shown in Figure 4E. The humanized 7A4 variant 7A4.v15 included selected fine-tuning positions in VL (position 68) and VH (positions 24 and 71) and the CDR-L3 change G94A, as shown in Figure 4B. 7G7 was humanized by grafting HVRs into the κ4 and subgroup I human common variable domains and the selected fine-tuning position (position 93) in VH, as shown in Figure 4C. This humanized variant is called 7G7.v1. 16F6 was humanized by transplanting the HVRs into the IGKV1-9*01 and IGHV4-59*01 human variable domains. The humanized variants contained a combination of different mouse fine-tuning positions, as shown in Figure 4E. The humanized 16F6 variant 16F6.v4 contained two changes in VL (I48L and F71Y) and selected fine-tuning positions in VL (positions 43 and 44) and VH (positions 71 and 78), as shown in Figure 4D.

Table 4: Light and heavy chain CDRs of humanized antibodies/Fabs

The affinity of the humanized variants as IgG for human and cynomolgus monkey TfR was determined by SPR (Figure 4E). The selected clones as Fab were also analyzed by SPR to evaluate the monovalent affinity (Table 7). In both cases, the SPR experiments were performed as described above.

Table 5: Biacore binding data for selected Fab-formatted variants

The binding epitope of the antibody was revalidated as follows. Tf-TfR blocking ELISA was performed in maxisorp plates coated with 2 μg/ml purified human TfR (in PBS) overnight at 4°C. The plates were washed with PBS/0.05% Tween 20 and blocked with Superblock blocking buffer (Thermo Scientific, Hudson, NH) in PBS. 50 μl of 12.5 μM human holo-Tf (R&D Systems, Minneapolis, MN) was added to the plates for 40 minutes. 50 μl of a titration of hu7A4.v15, hu15G11.v5, Tf competing antibody and hu7G7.v1 (starting at 10 ug/ml, 1:3 serial dilution) was added to the plates and incubated for 20 minutes. The plates were washed with PBS/0.05% Tween 20 and HRP-goat-anti-human Fcγ (Jackson ImmunoResearch) diluted 1:1000 was added to the plates and incubated for 1 hour at room temperature. The plates were washed with PBS/0.05% Tween 20 and detected with TMB substrate (BioFX Laboratories, Owings Mills).

The HFE-TfR binding ELISA was performed in maxisorp plates coated with 1 μg/ml HFE (in PBS) overnight at 4°C. The plates were washed with PBS/0.05% Tween 20 and blocked with Superblock blocking buffer (Thermo Scientific, Hudson, NH) in PBS. A titration of human TfR was added to the plates (starting at 100 μg/ml, serially diluted 1:3) and incubated for 1 hour. 1 μg/ml of hu15G11.v5, hu7A4.v15, or hu7G7.v1 was then added to the plates for 1 hour. The plates were washed with PBS/0.05% Tween 20, and a 1:1000 dilution of HRP-goat-anti-human Fcγ (Jackson ImmunoResearch) was added to the plates and incubated for 1 hour at room temperature. Plates were washed with PBS/0.05% Tween 20 and probed with TMB substrate (BioFX Laboratories, Owings Mills). An HFE-TfR blocking ELISA was performed in maxisorp plates coated with 1 μg/ml HFE in PBS overnight at 4°C. Plates were washed with PBS/0.05% Tween 20 and blocked with Superblock blocking buffer in PBS (Thermo Scientific, Hudson, NH). In NUNC ^™ plates, titrations of hu7A4.v15, hu15G11.v5, Tf competitor antibodies, human holo-Tf, and control IgG (400 μg for all antibodies, 8000 μg/ml for holo-transferrin, serially diluted 1:3) were combined with 2 μg/ml biotinylated human TfR and incubated for 1 hour. The mixture was then added to the HFE-coated plates and incubated at room temperature for 1 hour. The plate was washed with PBS/0.05% Tween 20, and HRP-streptavidin (SouthernBiotech, Birmingham) diluted 1:1000 was added to the plate and incubated at room temperature for 1 hour. The plate was washed with PBS/0.05% Tween 20, and biotinylated human TfR bound to the plate was detected using TMB substrate (BioFX Laboratories, Owings Mills).

The binding of these humanized variants to TfR was not affected by the presence of 6.3 μM holo-Tf, while the binding of Tf competing antibodies that bind to the Tf binding site on TfR was inhibited ( FIG5 ). In addition, humanized 7A4.v15, 15G11.v5, and 7G7.v1 were still able to bind to HFE-captured huTfR, indicating that they did not affect the binding of huTfR to immobilized HFE ( FIG6A ). In a related experiment, 7A4.v15 and 15G11.v5 did not block the binding of biotinylated TfR to immobilized HFE. In contrast, this interaction was blocked by Tf competing antibodies and holo Tf ( FIG6B ). HFE and Tf are known to share similar epitopes on TfR (Bennet et al., Nature (2000) 403, 46-53).

Immobilized 15G11v.5 and anti-TfR ^C12 were evaluated for binding to biotinylated human TfR ECD or monovalent M13 phage displaying the human TfR apical domain. Anti-TfR ^C12 was derived from a synthetic phage library that was panned against human TfR ECD and competes with transferrin for binding to sites on human TfR. The antibodies were coated onto Maxisorp plates at 1 μg/ml (in PBS). Bound biotinylated human TfR ECD or TfR-apical domain phage were detected using HRP-streptavidin (GE healthcare, RPN 4401V) or HRP-anti-M13 (GE healthcare, 27-9421-01), respectively. Figure 25 shows that 15G11v.5 binds to the human TfR apical domain. The 15G11v.5 binding site is mapped in the apical domain, which is a site away from the TfR ligand binding site.

Example 2: Affinity Engineering of Human/Cynomolgus Monkey Cross-Reactive Anti-TFR Antibodies

In addition to the humanized variants described above, additional affinity-engineered variants were prepared. Exemplified herein are affinity modifications of 15G11.v5 and 7A4.v15. Affinity variants were generated by making single alanine substitutions in CDR-L3 or CDR-H3 using standard techniques. These variants were screened as IgGs by ELISA and SPR to identify positions important for binding to human and cynomolgus monkey TfR; the monovalent affinity of selected variants as Fabs was also determined. Ala-scanning variant IgGs or Fabs were expressed in 293 cells, and binding to human or cynomolgus monkey TfR was quantified by ELISA in maxisorp plates coated overnight at 4°C with 1.8 μg/ml goat anti-human Fcγ (Jackson ImmunoResearch) (in PBS). The plates were washed with PBS/0.05% Tween 20 and blocked with Superblock blocking buffer (Thermo Scientific, Hudson, NH) in PBS. The supernatant containing expressed IgG was serially diluted 1:5 and added to the plate for 1 hour. Purified hu15G11.v5 or hu7A4.v15 was used as a standard (1:5 dilution starting with 1 ug/ml). The plate was washed with PBS/0.05% Tween 20, and a 1:1000 dilution of HRP-goat-anti-κ (Southern Biotech) was added to the plate and incubated at room temperature for 1 hour. The plate was washed with PBS/0.05% Tween 20 and detected with TMB substrate (BioFX Laboratories, Owings Mills). Binding was also determined by SPR, as described above. The results are shown in Figures 7A (15G11.v5 variant) and 7B (7A4.v15 variant).

Additional variants of 15G11.v5 with single alanine substitutions at positions in CDR-L1, CDR-L2, CDR-H1, and CDR-H2 were also generated, expressed, and initially screened for binding to human and cynomolgus monkey TfR by ELISA (Table 6). Hu/Cy binding ELISAs were performed in maxisorp plates coated overnight at 4°C with 2 μg/ml purified human or cynomolgus monkey TfR (in PBS). The plates were washed with PBS/0.05% Tween 20 and blocked with Superblock blocking buffer (Thermo Scientific, Hudson, NH) in PBS. Cell culture supernatants containing expressed Ala-scan variant IgGs were serially diluted 1:5 and added to the wells for 1 hour. The plates were washed with PBS/0.05% Tween 20, and a 1:1000 dilution of HRP-goat-anti-human Fcγ (Jackson ImmunoResearch) was added to the plates and incubated for 1 hour at room temperature. Plates were washed with PBS/0.05% Tween 20 and detected with TMB substrate (BioFX Laboratories, Owings Mills).

Table 6: ELISA analysis of hu15G11.v5 IgG Ala variants

Selected variants were then purified and their monovalent affinity to human or cynomolgus monkey TfR was determined by SPR (Table 7).

Table 7: Monovalent SPR analysis of selected 15G11.v5 Fab alanine variants

Example 3: Construction and in vitro analysis of bispecific anti-human TfR antibodies

Certain of the aforementioned antibody variants were reformatted into bispecific antibodies with a second arm that specifically binds to BACE1 using the 'knobs-in-holes' bispecific antibody construction technology (Carter, P. (2001) J. Immunol. Methods 248, 7-15; Ridgway, J.B., Presta, L.G., and Carter, P. (1996) Protein Eng. 9, 617-621; Merchant, A.M., Zhu, Z., Yuan, J.Q., Goddard, A., Adams, C.W., Presta, L.G., and Carter, P. (1998) Nat. Biotechnol. 16, 677-681; Atwell, S., Ridgway, J.B., Wells, J.A., and Carter, P. (1997) J. Mol. Biol. 270, 26-35). Anti-human TfR antibodies Hu15G11.v5, Hu15G11.LC92A, Hu15G11.HC52A, and Hu15G11.HC53A were used to engineer the TfR binding arm of a bispecific antibody. For anti-TfR (hole) and anti-BACE1 (knob), in addition to the knob and hole mutations in the Fc region, all half-antibodies contained a mutation (N297G) that abolished effector function in the Fc region, and Hu15G11.v5 and Hu15G11.LC92A contained an additional Fc mutation (D265A) that abolished effector function. Knob and hole half-antibodies were purified separately from E. coli and combined at an anti-TfR ratio of 1:1.1 to prevent the formation of anti-TfR homodimers. The assembling of bispecific antibodies was completed by reductive annealing at room temperature for at least three days in a buffer solution (pH 8.0) containing reduced glutathione and 200mM arginine at a 100x ratio with the antibody. After assembly, the bispecific antibodies were purified by hydrophobic interaction chromatography. Assembly was verified by liquid chromatography-mass spectrometry and SDS-PAGE. The purified antibodies were confirmed to be homogeneous and monodisperse by size exclusion and multi-angle laser spectroscopy.

The resulting bispecific antibodies were designated 15G11.v5 (anti-TfR ¹ ), 15G11.W92A (15G11.LC92A or anti-TfR ² ), Hu15G11.N52A (anti-TfR ^52A ), and Hu15G11.T53A (anti-TfR ^53A ). The monovalent affinity and kinetics of 115G11.v5 and 115G11.W92A for human and cynomolgus monkey TfR were determined by SPR as described above (see Table 9). Anti-TfR ¹ and anti-TfR ² have similar monovalent binding affinities to mouse TfR as anti- ^TfRA and anti- ^TfRD (see Atwal et al., Sci. Transl. Med. 3, 84ra43 (2011); Yu et al., Sci. Transl. Med. 25 May 2011: Vol. 3, Issue 84, p. 84ra44).

Table 8: Monovalent SPR analysis of 15G11.v5 (TfR ¹ ) and 15G11.W92A (LC92A, TfR ² )

In addition, as described previously, the binding affinities of anti- ^TfR1 , anti- ^TfR2 , Hu15G11.N52A, and Hu15G11.T53A bispecific antibodies to human and cynomolgus monkey TfR were measured by SPR. As shown in Table 9 below, anti- ^TfR52A and anti- ^TfR53A had binding affinities to human and cynomolgus monkey TfR between TfR1 ^h15G11.v5 and TfR2 ^LC92A .

Table 9

Example 4: Effects of effector-containing and effector-free monospecific and bispecific antibodies on human erythroleukemia cell lines and primary bone marrow mononuclear cells

Previous studies in mice have established that antibodies that bind to murine TfR and possess effector function and/or complement-binding capacity selectively deplete TfR-expressing reticulocytes. To determine whether the depletion observed in the mouse studies was specific to the murine system, further experiments were performed using anti-TfR that binds to human TfR.

Peripheral blood mononuclear cells (PBMCs) from healthy donors were used as effector cells to carry out ADCC assays. Human erythroleukemia cell lines (HEL, ATCC) and primary human bone marrow mononuclear cells (AllCells, Inc.) were used as target cells. In order to minimize the inter-donor variability that may be caused by the allotype differences at residue 158 position in FcγRIIIA, in the first group of experiments (Fig. 8A-B), donors were restricted to those carrying heterozygous RcγRIIIA genotype (F/V158). For the second group of experiments (Fig. 9A-B), only HEL cells were used as target cells, and PBMCs were from healthy donors carrying F/V158 genotype or FcγRIIIA V/V158 genotype. Due to its known association with increased NK cell-mediated ADCC activity and ability to bind IgG4 antibodies (Bowles and Weiner, 2005; Bruhns et al. 2008), the V/V158 genotype was also included in this assay. Cells were counted and viability was determined by (Beckman Coulter; Fullerton, CA) according to the supplier's instructions.

PBMCs were isolated by density gradient centrifugation using Uni-Sep ^™ blood separation tubes (Accurate Chemical & Scientific Corp.; Westbury, NY). Target cells were seeded at 4×10 ⁴ /well in 96-well round-bottom plates in 50 μL of assay medium (RPMI-1640 with 1% BSA and 100 units/mL penicillin and streptomycin). Serial dilutions of test and control antibodies (50 μL/well) were added to the plates containing target cells and then incubated at 37°C, 5% CO ₂ for 30 minutes to allow opsonization. The final concentration of the antibodies ranged from 0.0051-10,000 ng/mL, following a 5-fold serial dilution for a total of 10 data points. After incubation, 1.0×10 ⁶ PBMC effector cells in 100 μL of assay medium were added to each well to produce an effector:target cell ratio of 25:1, and the plates were incubated for an additional 4 hours. At the end of the incubation, the plates were centrifuged and the supernatants were assayed for lactate dehydrogenase (LDH) activity using a Cytotoxicity Detection Kit ^™ (Roche Applied Science; Indianapolis, IN). The LDH reaction mixture was added to the supernatant and the plates were incubated at room temperature for 15 minutes with constant shaking. The reaction was terminated with 1 M H ₃ PO ₄ and the absorbance was measured at 490 nm using a SpectraMax Plus microplate reader (background measured at 650 nm was subtracted for each well). The absorbance of wells containing only target cells served as a control for background (low control), while wells containing target cells lysed with Triton-X100 provided the maximum signal available (high control). Antibody-independent cellular cytotoxicity (AICC) was measured in wells containing target cells and effector cells without the addition of antibody. The degree of specific ADCC was calculated as follows:

ADCC of sample dilutions was plotted against antibody concentration, and dose-response curves were fit to a four-parameter model using SofiMax Pro.

In the first group of experiments, human erythroleukemia cell lines (HEL cells) or primary human bone marrow mononuclear cells were used as target cells to assess the ADCC activity of various anti-human TfR constructs. In ADCC assays, anti-gD WT IgG1 was used as a negative control, mouse anti-human HLA (category 1) was used as a positive control, and the anti-human TfR1 antibody 15G11 with bivalent IgG1 effector function was detected at different concentrations, and the bispecific form of the antibody, which has the anti-BACE1 arm of human IgG1 form, comprising D265A and N297G mutations, thereby eliminating effector function (see Example 3). The results are shown in Figures 8 A and 8B. Using HEL cells as targets (Figure 8 A) or bone marrow mononuclear cells as targets (Figure 8 B), the monospecific, effector-positive anti-human TfR antibody 15G11 triggered significant ADCC activity. This activity was similar to that of the positive control anti-human HLA antibody on HEL cells and was at a robust but lower level relative to the positive control activity on bone marrow mononuclear cells. The slightly lower levels observed in the bone marrow mononuclear cell experiments may be due to the fact that only a portion of the heterogeneous mixture of myeloid and erythroid lineage PBMC cells used in the experiment expressed high levels of TfR, while HEL cells had consistently high TfR expression throughout the clonal cell population. In sharp contrast, the bispecific effector-free anti-human TfR/BACE1 antibody did not exhibit any ADCC activity in either HEL or bone marrow mononuclear cells, which was similar to the negative control.

In a second set of experiments, the effect of switching antibody isotypes in this assay system was determined. The ADCC assay steps were the same as described above, except that all target cells were HEL cells and effector cells were PBMCs from healthy human blood donors carrying either a heterozygous FcγRIIIa-V/F158 genotype or a homozygous FcγRIIIa-V/V158 genotype. All anti-human TfRs tested were bispecific, with anti-gD on three different Ig backbones: wild-type human IgG1, human IgG1 with the N297G mutation, and human IgG4. Anti-Aβ antibodies with a human IgG4 backbone were also tested, and mouse anti-human HLA (class I) served as a positive control. The results are shown in Figures 9A and 9B. As predicted based on the correlation between effector cell activation and V/V158 genotype (Bowles and Weiner 2005), PBMCs from V/V158 donors more robustly elicited ADCC activity (affecting ~45% of target cells) compared to F/V158 donors (affecting ~25% of target cells) (compare Figure 9A with Figure 9B). Anti-TfR/gD with wild-type IgG1 induced robust ADCC in HEL cells, while anti-TfR/gD without effector IgG1 did not exhibit any ADCC activity in HEL cells, replicating the results of the first set of experiments. Notably, anti-TfR/gD with the IgG4 isotype exhibited slight ADCC activity at concentrations of 100 ng/mL or higher. This activity was not observed in the anti-Aβ IgG4 results, indicating that ADCC activity requires TfR binding. This finding correlates with previous reports that IgG4 has minimal but measurable effector function (Adolffson et al., J. Neurosci. 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)).

Example 5: In vivo evaluation of bispecific anti-human TfR/BACE1 bispecific antibodies

A. Pharmacokinetics, pharmacodynamics and safety studies

To evaluate the drug concentration, pharmacodynamics, and safety of bispecific anti-human TfR antibodies in vivo, cynomolgus monkeys (Macaca fascicularis) were administered bispecific antibodies using anti-TfR antibody clone 15G11 (anti-TfR ¹ / BACE1) paired with the same anti-BACE1 arm as used in the previous examples, or clone 15G11.LC92A (anti-TfR ² / BACE1) paired with the same anti-BACE1 arm as used in the previous examples, or Hu15G11.N52A (anti-TfR ^52A / BACE1) and Hu15G11.T ^53A (anti-TfR ^53A / BACE1). These bispecific antibodies are in human IgG1 format with N297G or D265A and N297G mutations that abolish effector function, as described previously. As a control, anti-gD molecules on human IgG1 were used. Since the cross-reactivity of these anti-TfR antibodies is limited to non-human primates and humans, this study was conducted in non-human primates. In addition, studies have shown that the drug transport mechanism between cerebrospinal fluid (CSF) and plasma compartments may be similar between humans and primates (Poplack et al., 1977). The antibody was administered to conscious cynomolgus monkeys in the saphenous vein by a single intravenous (IV) bolus injection at a dose of 30 mg/kg using a cistern magna indwelling catheter. At different time points up to 60 days after administration, plasma, serum and (CSF) were sampled. Sample analysis included hematology (whole blood), clinical chemistry (serum), antibody concentration (serum and CSF) and pharmacodynamic response (plasma and CSF) to the antibody. See Figure 10 for detailed sampling scheme.

The concentration of the administered antibody in cynomolgus monkey serum and CSF was measured by ELISA using a sheep anti-human IgG monkey-adsorbed antibody coating followed by the addition of serum samples starting at a 1:100 dilution and completed by the addition of a goat anti-human IgG antibody conjugated to monkey-adsorbed horseradish peroxidase for detection. The assay had a standard curve range of 0.78-50 ng/mL and a limit of detection of 0.08 μg/mL. Results below this limit of detection were reported as less than reportable results (LTR).

Figure 11A-B shows the result of the pharmacokinetic analysis of anti-TfR1/BACE1 and anti-TfR2/BACE1.Prediction anti-gD pharmacokinetic pattern is about the typical human IgG1 antibody in cynomolgus monkey, and average clearance is 3.98mL/ day/kg.May be due to the removal of surrounding target-mediation, the removal of two anti-TfR/BACE1 antibodies is faster than anti-gD.Anti-TfR1/BACE1 has the fastest clearance rate, which is consistent with its highest binding affinity for TfR, and compared with anti-TfR1/BACE1, anti-TfR2/BACE1 shows improved pharmacokinetic pattern (that is, extended exposure in serum), which may be due to its reduced affinity for TfR causing.The clearance rates of anti-TfR1/BACE1 and anti-TfR2/BACE1 are respectively 18.9mL/ day/kg and 8.14mL/ day/kg. All antibodies were detected in CSF at concentrations one thousandth of the serum concentration. However, there was a high degree of variability in CSF antibody concentrations between molecules, and overall no detectable differences.

Table 10: Mean (±SD) PK parameter estimates for all tested antibodies following a single IV bolus dose of 30 mg/kg in cynomolgus monkeys (n=5)

SD = standard deviation; IV = intravenous; _AUCall = area under the concentration-time curve from time 0 to the time of the last measurable concentration; _AUCinf = area under the concentration-time curve extrapolated to infinity; _Cmax = maximum observed serum concentration; CL = clearance; _Vss = volume of distribution at steady state; Min = minimum; Max = maximum.

Figure 19 shows the result of the pharmacokinetic analysis about anti-TfR ¹ /BACE1, anti-TfR ^52A /BACE1 and anti-TfR ^53A /BACE1.It may be due to the clearance rate of surrounding target-mediation, and the clearance of all anti-TfR/BACE1 antibodies is faster than anti-gD.Anti-TfR ¹ /BACE1 has the fastest clearance rate, which is consistent with its highest binding affinity for TfR, and compared with anti-TfR ¹ /BACE1, anti-TfR ^52A /BACE1 and anti-TfR ^53A /BACE1 show improved pharmacokinetic pattern (that is, extended exposure in serum), which may be due to the affinity for TfR reduced by anti-TfR ^52A /BACE1 and anti-TfR ^53A /BACE1.

In order to observe the pharmacodynamic effect of response anti-TfR/BACE1 administration, we measure A β _1-40 and sAPP α and sAPP β levels in cynomolgus monkey plasma and CSF. Using anti-A β _1-40 specific polyclonal antibody coating, sample is then added and completed by adding mouse anti-human A β _1-40 monoclonal antibody conjugated to horseradish peroxidase for detection, A β _1-40 is measured with ELISA. The assay has a plasma detection limit of 60pg/mL and a CSF detection limit of 140pg/mL. Results below this concentration are reported as less than reportable (LTR) results. The CSF concentrations of sAPP α and sAPP β are determined using sAPP α/sAPP β multi-point assay (Mesoscale Discovery (Gaithersburg, MD)). CSF was thawed on ice and diluted 1:10 into 1% BSA in TBS-T (10 mM Tris buffer, pH 8.0, 150 mM NaCl, 0.1% Tween-20). The assay was performed according to the supplier's protocol and had a lower limit of quantification of 0.05 ng/ml for sAPPα and 0.03 ng/mL for sAPPβ.

Figures 12A-E summarize the pharmacodynamic behavior of the antibodies. In the periphery, plasma Aβ _1-40 levels remained unchanged after anti-gD administration, but decreased transiently after anti-TfR/BACE1 administration. Both variants reduced plasma Aβ _1-40 levels, with 50% maximum inhibition achieved one day after dosing. Plasma Aβ _1-40 levels gradually recovered, with animals given anti-TfR ¹ /BACE1 returning to baseline Aβ _1-40 levels approximately 14 days after dosing. In animals treated with anti-TfR ² /BACE1, Aβ _1-40 levels returned to baseline levels 21-30 days after dosing. Both anti-TfR/BACE1 antibodies reduced CSF Aβ _1-40 levels, with no changes observed in animals given anti-gD. Compared with anti-TfR ² / BACE1 (average maximum inhibition of 20% of baseline), anti-TfR ¹ / BACE1 administration resulted in a more significant reduction in CSF Aβ _1-40 levels (average maximum inhibition of 50% of baseline). In animals treated with anti-TfR/BACE1, the production of sAPPβ was inhibited, but not inhibited in animals receiving anti-gD. Similar to the results on Aβ40, the inhibitory effect of anti-TfR ¹ / BACE1 on the production of sAPPβ was stronger than that of anti-TfR ² / BACE1. During BACE1 inhibition, both anti-TfR ¹ / BACE1 and anti-TfR ² / BACE1 stimulated the production of sAPPα, and the response was negatively correlated with the inhibition levels observed for sAPPβ and Aβ _1-40 . SAPPα and sAPPβ are the main processed products of amyloid precursor protein (APP), and their levels are highly correlated. The sAPPβ/sAPPα ratio normalizes the results for potential changes in basal APP expression during the study or potential pre-analytical differences in CSF collection and processing. The CSF sAPPβ/sAPPα ratio using anti- ^TfR1 /BACE1 demonstrated a more robust PD effect than anti- ^TfR2 /BACE1. Therefore, these results support target engagement of the anti-TfR/BACE1 antibody, namely BACE1.

The PD responses of anti- ^TfR52A /BACE1 and anti- ^TfR53A /BACE1 were also related to the duration of antibody exposure, and the reduced affinity TfR arm showed increased _Aβ40 reduction (data not shown). These data also support the binding of these bispecific antibodies to the target.

Taken together, these results suggest that bispecific anti-TfR/BACE1 antibodies with affinity for human TfR between anti-TfR ¹ /BACE1 and anti-TfR ² /BACE1 may have a desirable pharmacokinetic/pharmacodynamic balance.

No safety signals were observed in this study. There were no significant effects on any hematological or clinical chemistry parameters in monkeys administered 30 mg/kg of any bispecific antibody up to 60 days after dosing. Importantly, since these antibodies are effector-function impaired and in normal primates, the overall level of circulating early reticulocytes with high TfR levels is very low (see Example 4), as predicted, reticulocyte levels were not affected by treatment with anti-TfR ¹ /BACE1 or anti-TfR ² /BACE1 (Figure 13).

Example 6: In vivo evaluation of bispecific anti-human TfR/BACE1 bispecific antibodies

In order to examine the relationship between the pharmacodynamics of antibodies in CSF and the pharmacokinetics in the brain, cynomolgus monkeys (Macaca fascicularis) were administered with bispecific antibodies anti-TfR ¹ /BACE1 or anti-TfR ² /BACE1, as described in the preceding examples. These bispecific antibodies adopt the human IgG1 format, with D265A and N297G mutations that eliminate effector function. Anti-gD molecules on human IgG1 were used as controls. For comparison, we also administered bivalent anti-BACE1 antibodies, which were the same clones used for bispecific antibodies. Antibodies were administered to conscious cynomolgus monkeys saphenous veins using a cisterna magna indwelling catheter via a single intravenous (IV) bolus injection at a dose of 30 mg/kg. Baseline CSF samples were collected 24 and 48 hours before administration, and another CSF sample (as illustrated in Figure 14) was collected 24 hours after administration. After 24 hours of CSF collection, animals were perfused with saline, and brains were collected for antibody concentration analysis. Different brain regions were homogenized in 1% NP-40 (Cal-Biochem) in PBS containing Complete Mini EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics). The homogenized brain samples were rotated at 4°C for 1 hour and then centrifuged at 14,000 rpm for 20 minutes. The supernatant was isolated and used for brain antibody measurement using the ELISA method described in the previous example. Blood was also collected to verify peripheral exposure and pharmacodynamic response, which was similar to our observations in Example 5.

The pharmacodynamic effects of anti-TfR ¹ / BACE1 and anti-TfR ² / BACE1 evaluated in CSF were also similar to those observed in the previous examples. Figure 15 demonstrates that after administration with anti-TfR ¹ / BACE1, the CSF sAPPβ / sAPPα ratio is robustly reduced. In this study, anti-TfR ² / BACE1 did not show a significant reduction 24 hours after administration. Anti-BACE1 also showed no effect. Analysis of antibody concentrations in the brain revealed that both control IgG and anti-BACE1 antibodies had limited uptake into the brain, with uptake levels just above the detection level of our assay (average ~ 670pM). Anti-TfR ² / BACE1 had ~ 3-fold improved brain uptake compared to control IgG (average ~ 2nM), and anti-TfR ¹ / BACE1 had the best brain uptake, ~ 15-fold greater than control IgG (average ~ 10nM). In our study, brain antibody concentrations for the different antibodies correlated with the pharmacodynamic responses observed in CSF, with anti- ^TfR1 /BACE1 having the best brain uptake and the most robust pharmacodynamic effects, and anti- ^TfR2 /BACE1 having less brain uptake and more moderate effects.

These results extend our previous findings to prove that the bispecific antibodies of TfR-binding improve the intake in non-human primates. In primates, as in mice, there may be an optimal affinity for TfR that best balances brain uptake and TfR-mediated removal. In our experiments, higher affinity anti-TfR ¹ /BACE1 showed good brain uptake and was affected by the removal of peripheral targets-mediation. TfR ² /BACE1 with reduced affinity has improved clearance characteristics, but seems to have such low TfR binding that it cannot be effectively transported by TfR (mainly in the same way, the lowest affinity anti-TfR antibody TfR ^E in US2012/0171120 has some affinity thresholds, above which affinity is too low to allow sufficient interaction between antibody and TfR, so that the antibody will remain associated with TfR when TfR starts the translocation process). From the results of this experiment, it is predicted that anti-human/cynomolgus TfR/BACE1 bispecific antibodies with an affinity for TfR between ^{TfR 1} and TfR ² will have improved uptake and clearance properties in this system compared to anti-TfR ¹ /BACE1 or anti-TfR 2 /BACE1.

Example 7: Generation of additional null effector mutations in the context of bispecific transferrin receptor antibodies

Other mutations in the Fc region that abolish effector function, in addition to N297G and D265A, were examined for their ability to reduce or prevent the depletion of TfR-expressing reticulocytes. Specifically, the Fc mutations L234A, L235A, and P329G ("LALAPG") described in U.S. Application Publication No. 2012/0251531 (which is incorporated herein by reference) were combined in an anti-TfR ^D /BACE1 antibody (which is described in International Application Publication No. WO 2013/177062, which is incorporated herein by reference in its entirety).

Pharmacokinetic analysis and reticulocyte counts after single antibody administration in mice were performed as follows. All studies used wild-type C57B/6 female mice aged 6-8 weeks. Animal care was performed in accordance with institutional guidelines. Mice were intravenously administered with a single 50 mg/kg dose of anti-gD antibody (mouse IgG2a) with LALAPG mutation and anti-TfR ^D /BACE1 antibody (rat/mouse chimera) with LALAPG mutation. The total injection volume did not exceed 250 μL, and when necessary, the antibody was diluted into D-PBS (Invitrogen). After 24 hours, whole blood was collected in EDTA microtubes (BD Diagnostics) before perfusion, allowed to stand at room temperature for 30 minutes, and centrifuged at 5000 x g for 10 minutes. The top layer of plasma was transferred to a new tube for antibody measurement.

Total antibody concentrations in mouse plasma were measured using an anti-mouse IgG2a (allotype a)/anti-mouse IgG2a (allotype a) ELISA. NUNC 384-well Maxisorp immunoplates (Neptune, NJ) were coated overnight at 4°C with mouse anti-mouse IgG2a allotype A (an allotype A-specific antibody) (BD/Pharmigen San Jose, CA), and the plates were blocked with PBS, 0.5% BSA at 25°C for 1 hour. Each antibody (anti-gD and anti-TfR/BACE1 bispecific variant) was used as a standard to quantify the concentration of each antibody. The plates were washed with PBS, 0.05% Tween-20 using a microplate washer (Bio-Tek Instruments, Inc., Winooski, VT) and the standards and samples diluted in PBS containing 0.5% BSA, 0.35 M NaCl, 0.25% CHAPS, 5 mM EDTA, 0.2% BgG, 0.05% Tween-20, and 15 ppm (Sigma-Aldrich) were added and incubated at 25°C for 2 hours. Bound antibodies were detected with biotin-conjugated mouse anti-mouse IgG2a allotype A (an antibody specific for allotype A) (BD/Pharmigen San Jose, CA). Bound biotin-conjugated antibodies were detected with horseradish peroxidase-conjugated streptavidin (GE Healthcare Life Sciences, Pittsburgh, PA). Samples were developed with 3,3',5,5'-tetramethylbenzidine (TMB) (KPL, Inc., Gaithersburg, MD) and absorbance was measured at 450 nm on a Multiskan Ascent reader (Thermo Scientific, Hudson, NH). Concentrations were determined from a standard curve using a four-parameter nonlinear regression program. The assay had a lower limit of quantification (LLOQ) value of 78.13 ng/ml in plasma. Statistical analysis of differences between experimental groups was performed using a two-tailed unpaired t-test.

When administered with an anti- ^TfRD /BACE1 antibody containing the FcLALAPG mutation, mice did not exhibit clinical symptoms as previously observed with antibodies with full effector function. See Couch et al., Sci. Trans. Med. 5:183ra57 (2013). Figure 20 shows the results of pharmacokinetic analysis.

In addition, immature and total reticulocyte counts were determined using a Sysmex XT2000iV (Sysmex, Kobe, Japan) according to the manufacturer's instructions. No differences were observed in the immature reticulocyte fraction or total reticulocyte counts 24 hours after dosing using any of the antibodies tested, as shown in Figure 21. These results suggest that the LALAPG mutation not only abolishes antibody effector function, but also reduces complement fixation and complement-mediated reticulocyte clearance observed even with the effector-free antibody framework (Couch et al. 2013). This is consistent with another report that combining the LALA mutation on human IgG1 can limit complement fixation (Hessell et al., Nature 449: 101-104 (2007)).

Example 8 - Generation of FcRn ^HIGH bispecific variants

To increase the half-life of bispecific antibodies, and thereby potentially increase the concentration of the antibody in the brain, bispecific variants were prepared that contained mutations in the IgG constant domain, and specifically in the Fc receptor-neoantibody (FcRn) binding domain (FcRn ^HIGH mutation). The FcRn binding domain has been implicated in maternal-fetal transfer of antibodies. See Story et al., J. Exp. Med., 180: 2377-2381, 1994. Amino acid substitutions in the FcRn binding domain increase the affinity of the constant domain for FcRn, thereby increasing the half-life of the antibody.

The FcRn binding domain mutations M252Y, S254T, and T256E (YTE) have been described to increase FcRn binding and, therefore, increase the half-life of the antibody. See U.S. Published Patent Application No. 2003/0190311 and Dall'Acqua et al., J. Biol. Chem. 281: 23514-23524 (2006). Additionally, the FcRn binding domain mutations N434A and Y436I (AI) have been described to increase FcRn binding. See Yeung et al., J. Immunol. 182: 7663-7671 (2009). The YTE (M252Y/S254T/T256E) and AI (N434/Y436I) mutations were incorporated into anti-TfR ^52A / BACE1 and anti-TfR ² / BACE1 bispecific antibodies containing either WT human IgG1 or the effector-less LALAPG or N297G mutations. Additionally, an FcRn ^HIGH mutation was generated in an anti-gD hIgG1 antibody as a control. Mutations were constructed using Kunkel mutagenesis, antibodies were transiently expressed in CHO cells, and proteins were purified using protein A chromatography followed by size exclusion chromatography (SEC).

The binding of FcRn ^HIGH variant antibodies to FcRn was measured using BIAcore. Human and cynomolgus monkey FcRn proteins were expressed in CHO cells and purified using IgG affinity chromatography. Data were obtained on a BIAcore T200 instrument. A series S sensor chip CM5 (GE Healthcare, Cat. BR100530) was activated with EDC and NHS reagents according to the supplier's instructions, and coupled with an anti-Fab antibody (human Fab capture kit, GE Health care Bio-science. AB SE-75184, Upsala, Sweden) to obtain approximately 10,000 response units (RU), and unreacted groups were then blocked with 1 M ethanolamine. For affinity measurement, antibodies were first injected at a flow rate of 10 μl/min to capture approximately 1000 RU on three different flow cells (FC), except for FC1 (reference), and then a 2-fold serial dilution of human FcRn (or cynomolgus monkey FcRn) in pH 6 buffer (0.1 M sodium phosphate) was injected (flow rate: 30 μl/min), one after another from low (1 nM) to high (25 μM) in the same cycle, without regeneration between injections. Sensograms were recorded and the reference and buffer were subtracted before evaluation with BIAcore T200 evaluation software (version 2.0). Affinity was determined by analyzing the binding level of the steady state based on a 1: 1 binding model. The binding affinities of LALAPG, N297G, LALAPG.YTE and LALAPG.AI variants of anti-TfR ^52A / BACE1 are shown in Table 11 below. The data show that the FcRn ^HIGH variant has increased affinity for human and cynomolgus FcRn in endosomes (pH 6).

Table 11

Selected FcRnHIGH variants were tested in cynomolgus monkeys to determine whether enhanced FcRn affinity could improve pharmacokinetic properties and/or increase brain exposure of anti-TfR/BACE1 antibodies.

To evaluate the safety of the FcRn ^HIGH mutation and the effector, certain bispecific antibodies were administered to mice expressing human transferrin receptor knock-in. The huTfR knock-in mice were generated as follows. A construct for targeting human TFRC cDNA to the C57BL/6 Tfrc locus in ES cells was prepared using a combination of (Warming et al., Molecular and Cellular Biology vol. 26(18) pp. 6913-22 2006; Liu et al., Genome Research (2003) vol. 13(3) pp. 476-84) and standard molecular cloning techniques.

Briefly, Tfrc C57BL/6J BAC (RP23 BAC library) was modified by recombineering using a cassette flanking a short homolog of the mouse Tfrc gene (human TFRC cDNA, SV40 pA, and frt-PGK-em7-Neo-BGHpA-frt). The human TFRC cDNA cassette was inserted at the endogenous ATG and the remainder of Tfrc exon 2 plus the beginning of intron 2 were deleted. The targeted region in the BAC was then returned to pBlight-TK (Warming et al., Molecular and Cellular Biology, vol. 26 (18) pp. 6913-22 2006) along with flanking genomic Tfrc sequences as homology arms for ES cell targeting. Specifically, the 2950 bp 5' homology arm corresponds to (assembly NCBI37/mm9): chr.16: 32,610,333-32,613,282, and the 2599 bp 3' homology arm corresponds to chr.16: 32,613,320-32,615,918. The final vector was verified by DNA sequencing.

The Tfrc/TFRC KI vector was linearized with NotI and targeted to C57BL/6N C2 ES cells using standard methods (G418 positive and ganciclovir negative selection). Positive clones were identified using PCR and Taqman analysis, and verified by sequencing the modified locus. Correctly targeted ES cells were transfected with the Flpe plasmid to remove Neo, and then standard techniques were used to inject ES cells into blastocysts. After hybridization of the resulting chimera with C57BL/6N females, germline transmission was achieved.

Specifically, the antibodies listed in the table below were administered to huTfR knock-in mice at a single dose of 50 mg/kg, and 24 hours later, blood was drawn (and reticulocytes were counted). huIg1, N297G

Table 12

Antibody Isotype Number of mice Anti-gD huIg1, N297G 6 huIgG1, N297G 6 huIgG1, LALAPG 6 huIgG1, LALAPG/YTE 6 huIgG1, LALAPG/AI 6

After administration of anti-TfR ^52A / BACE1 LALAPG, LALAPG/YTE or LALAPG/AI antibodies (Groups 3-5 in Table 12), human TfR knock-in mice did not show clinical symptoms or reticulocyte loss (Figure 22) as previously observed using anti-TfR antibodies with full effector function (Couch et al. 2013). These results indicate that combining the LALAPG mutation with the human IgG1 construct also eliminates effector function and further suggest that the addition of the YTE or AI FcRn ^HIGH mutation does not interfere with the LALAPG mutation, giving the antibody the desirable properties of being effector-free.

ADCC assays were also performed to verify the effector-free state of LALAPG, LALAPG/YTE, and LALAPG/AI mutation combinations in human cell lines. As previously described, human erythroleukemia cell lines (HEL, ATCC) were used as target cells, and PBMCs were derived from healthy human blood donors carrying either the F/V158 genotype or the FcγRIIIA V/V158 genotype. Due to its known association with increased NK cell-mediated ADCC activity and its ability to bind IgG4 antibodies (Bowles and Weiner, 2005; Bruhns et al. 2008), the V/V158 genotype was also included in this assay. Cells were counted, and viability was determined by (BeckmanCoulter; Fullerton, CA) according to the supplier's instructions.

PBMCs were isolated by density gradient centrifugation using Uni-Sep ^™ blood separation tubes (Accurate Chemical & Scientific Corp.; Westbury, NY). Target cells were seeded at 4×10 ⁴ /well in 96-well round-bottom plates in 50 μL of assay medium (RPMI-1640 with 1% BSA and 100 units/mL penicillin and streptomycin). Serial dilutions of test and control antibodies (50 μL/well) were added to the plates containing target cells and then incubated at 37°C, 5% CO ₂ for 30 minutes to allow for opsonization. The final concentration of the antibodies ranged from 0.0051-10,000 ng/mL, following a 5-fold serial dilution for a total of 10 data points. After incubation, 1.0×10 ⁶ PBMC effector cells in 100 μL of assay medium were added to each well to produce an effector:target cell ratio of 25:1, and the plates were incubated for an additional 4 hours. At the end of the incubation, the plates were centrifuged and the supernatants were assayed for lactate dehydrogenase (LDH) activity using a Cytotoxicity Detection Kit ^™ (Roche Applied Science; Indianapolis, IN). The LDH reaction mixture was added to the supernatant and the plates were incubated at room temperature for 15 minutes with constant shaking. The reaction was terminated with 1 M H ₃ PO ₄ and the absorbance was measured at 490 nm using a SpectraMax Plus microplate reader (background measured at 650 nm was subtracted for each well). The absorbance of wells containing only target cells served as a control for background (low control), while wells containing target cells lysed with Triton-X100 provided the maximum signal available (high control). Antibody-independent cellular cytotoxicity (AICC) was measured in wells containing target cells and effector cells without the addition of antibody. The degree of specific ADCC was calculated as follows:

ADCC of sample dilutions was plotted against antibody concentration, and dose-response curves were fit to a four-parameter model using SoftMax Pro.

The results of the ADCC assay are shown in Figure 23. As expected, effector-positive anti-human TfR antibodies (anti-TfR ¹ / gD IgG1 WT) elicited significant ADCC activity on HEL cells. In contrast, anti-TfR ^52A / BACE1 antibody variants containing LALAPG, LALAPG/YTE, or LALAPG/AI mutations did not exhibit any ADCC activity in HEL cells, which was similar to the negative control anti-TfR ^52A / gD N297G antibody.

Although the foregoing invention has been described in some detail by way of illustration and example 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 patent and scientific literature cited herein are expressly incorporated herein by reference in their entirety.

Claims (47)

1. An antibody that binds to the isolated human transferrin receptor (TfR) and primate TfR, wherein the antibody does not inhibit the binding of transferrin to TfR, wherein the antibody comprises the HVR-H1 sequence of SEQ ID NO:53, the HVR-H2 sequence of SEQ ID NO:156, the HVR-H3 sequence of SEQ ID NO:55, the HVR-L1 sequence of SEQ ID NO:50, the HVR-L2 sequence of SEQ ID NO:51, and the HVR-L3 sequence of SEQ ID NO:52. 2. An antibody that binds to isolated human TfR and primate TfR, wherein the antibody does not inhibit the binding of transferrin to TfR, and wherein one or more properties of the antibody have been modified to reduce or eliminate the effect of the antibody on reticulocytes and/or reduce the severity or presence of acute clinical symptoms in a subject or mammal treated with the antibody, wherein the antibody comprises the HVR-H1 sequence of SEQ ID NO:53, the HVR-H2 sequence of SEQ ID NO:156, the HVR-H3 sequence of SEQ ID NO:55, the HVR-L1 sequence of SEQ ID NO:50, the HVR-L2 sequence of SEQ ID NO:51, and the HVR-L3 sequence of SEQ ID NO:52, wherein one or more properties are selected from the effector function of the Fc region of the antibody, the complement activation function of the antibody, the half-life of the antibody, and the affinity of the antibody for TfR. In contrast to the same type of wild-type antibody, the effector function or complement activation function has been reduced or eliminated. The effector function is reduced or eliminated by methods selected from: reducing the glycosylation of the antibody, modifying the antibody isotype to a naturally occurring isotype with reduced or eliminated effector function, and modifying the Fc region. The glycosylation of the antibody is reduced by methods selected from: preparing the antibody in an environment where wild-type glycosylation is not permitted; removing pre-existing carbohydrate groups from the antibody; and modifying the antibody to prevent wild-type glycosylation. The antibody is produced in a non-mammalian cell preparation system, or the antibody is synthesized in a specific manner. The Fc region of the antibody contains a mutation at position 297, which causes the wild-type asparagine residue at that position, according to the EU numbering system, to be replaced by another amino acid that interferes with the glycosylation at that position. The effector function is reduced or eliminated by at least one modification of the Fc region or CH1 structural domain. The effector function or complement activation function is reduced or eliminated by deleting all or part of the Fc region, or by modifying the antibody to exclude Fc or non-Fc region components with effector function or complement activation function. The modifications are selected from Fc region point mutations at the following positions according to the EU numbering system that weaken the binding to one or more Fc receptors: 234, 235, 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; weakening Fc region point mutations combined with C1q at the following positions according to EU numbering: 270, 322, 329 and 321; elimination of some or all of the Fc region, and point mutation at position 132 of the CH1 domain according to the EU numbering system. 3. The antibody of claim 1 or 2, comprising: (a) a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:153; (b) a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:105; or (c) the VH sequence in (a) and the VL sequence in (b). 4. The antibody of claim 3, comprising the following VH sequence: SEQ ID NO:153. 5. The antibody of claim 3, comprising the following VL sequence: SEQ ID NO:105. 6. An antibody comprising: the VH sequence of SEQ ID NO:153 and the VL sequence of SEQ ID NO:105. 7. The antibody of claim 2, wherein the modification is at least one point mutation in the Fc region selected from EU numbering systems 234, 235, 265, 297 and 329 that weakens the binding of the Fc region to one or more Fc receptors. 8. The antibody of claim 7, wherein the modification is at position 297 or 265 and 297 according to the EU numbering system. 9. The antibody of claim 7, wherein the modification is at positions 234, 235 and 329 according to the EU numbering system. 10. The antibody of claim 8, wherein the modification is N297G; D265A and N297A or D265A and N297G according to the EU numbering system. 11. The antibody of claim 8, wherein the modification is according to L234A, L235A and P329G of the EU numbering system. 12. The antibody of claim 2, wherein one or more of the characteristics are the half-life of the antibody. 13. The antibody of claim 12, wherein the half-life is increased by modification of the FcRn binding domain of the antibody at positions selected from 252, 254, 256, 434 and 436 according to the EU numbering system. 14. The antibody of claim 13, wherein the modification is at positions 252, 254 and 256 according to the EU numbering system. 15. The antibody of claim 13, wherein the modification is at positions 434 and 436 according to the EU numbering system. 16. The antibody of claim 14, wherein the modification is M252Y, S254T and T256E according to the EU numbering system. 17. The antibody of claim 15, wherein the modification is N434A and Y436I according to the EU numbering system. 18. The antibody of claim 2, conjugated to another compound that alleviates or helps reduce the effect on reticulocyte levels or acute clinical symptoms. 19. The antibody of claim 18, wherein the other compound protects reticulocytes from antibody-related consumption or supports the growth, development, or reconstruction of reticulocytes. 20. The antibody of claim 19, wherein the other compound is selected from erythropoietin (EPO), iron supplements, vitamin C, folic acid and vitamin B12, or erythrocytes or reticulocytes. 21. The antibody of claim 2, wherein the reduced affinity of the antibody for TfR is measured relative to a wild-type antibody of the same isotype that does not have reduced affinity for TfR. 22. The antibody of claim 1 or 2, wherein the antibody has a KD or IC50 against TfR of about 1 pM to about 100 μM. 23. The antibody of claim 1 or 2, wherein the antibody is conjugated to a therapeutic compound. 24. The antibody of claim 23, wherein the antibody is a multispecific antibody, and the therapeutic compound optionally forms part of the multispecific antibody. 25. The antibody of claim 24, wherein the multispecific antibody comprises a first antigen-binding site that binds to TfR and a second antigen-binding site that binds to brain antigens. 26. The antibody of claim 25, wherein the brain antigen is selected from the group consisting of: β-secretase 1 (BACE1), Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E (ApoE), α-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeating kinase 2 (LRRK2), perkinin, progerin 1, progerin 2, γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic protein receptor (p75NTR), and caspase 6. 27. The antibody of claim 26, wherein the multispecific antibody binds both TfR and BACE1. 28. The antibody of claim 26, wherein the multispecific antibody binds both TfR and Aβ. 29. The antibody of claim 23, wherein the therapeutic compound is a drug for a nervous system disease. 30. The antibody of any one of claims 1-2, wherein it is a monoclonal antibody. 31. The antibody of any one of claims 1-2, wherein it is a human antibody, a humanized antibody, or a chimeric antibody. 32. The antibody of any one of claims 1-2, wherein it is an antibody fragment that binds to human TfR and primate TfR. 33. The isolated nucleic acid encoding the antibody of any one of claims 1-32. 34. A host cell comprising the nucleic acid of claim 33. 35. A method for preparing an antibody, the method comprising culturing the host cell of claim 34 to produce the antibody, and optionally further comprising recovering the antibody from the host cell. 36. A pharmaceutical preparation comprising an antibody and a pharmaceutical carrier according to any one of claims 1-32. 37. Use of the antibody of any one of claims 1-32 in the preparation of a reagent for transporting one or more compounds across the BBB. 38. Use of the antibody of any one of claims 1-32 in the preparation of a medicament for treating nervous system diseases. 39. The application of claim 38, wherein the neurological disease is selected from the group consisting of: neurological disorders, neurodegenerative diseases, cancer, eye diseases, epileptic seizures, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders, and CNS inflammation. 40. Use of the antibody of any one of claims 1-32 in the preparation of a medicament for transporting one or more compounds across the BBB. 41. Use of the antibody of claim 23 in the preparation of a medicament for transporting a compound across the body's blood-brain barrier (BBB) of a subject, wherein the antibody transports the compound conjugated thereto across the BBB by exposure to the BBB. 42. Use of the antibody of claim 23 in the preparation of a medicament that increases the CNS exposure of a subject to a compound, wherein the antibody transports the compound conjugated thereto across the BBB by exposure to the BBB. 43. Use of the antibody of claim 23 in the preparation of a medicament that increases the retention of a compound administered to a subject in the CNS, wherein the antibody increases the retention of the compound in the CNS by exposure to the BBB. 44. Use of the antibody of claim 23 in the preparation of a medicament for treating nervous system diseases in mammals. 45. The application of claim 44, wherein the neurological disease is selected from the group consisting of: neurological disorders, neurodegenerative diseases, cancer, eye diseases, epileptic seizures, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders, and CNS inflammation. 46. The application of claim 42, wherein a therapeutic dose of the antibody conjugated with the compound is contained in the medicament. 47. The application of claim 46, wherein the therapeutic dose is TfR-saturated.