Bispecific antibodies and constructs for lysosomal targeted degradation and methods of use thereof

Abstract

Provided herein are antigen binding proteins (ABPs) that selectively bind to M6PR and its isoforms and homologs, and compositions comprising the ABPs. Also provided herein are bispecific antigen binding proteins (ABPs) that selectively bind to an internalizing receptor and a soluble or cell surface target molecule and its isoforms and homologs, and compositions comprising the ABPs.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 397,751, filed August 12, 2022, which is incorporated herein by reference in its entirety.

[0002] 2. Sequence Listing This application contains a Sequence Listing that has been submitted through the Patent Center and is incorporated herein by reference in its entirety. The XML copy was created on XXXX, is named XXXX, and is XXX bytes in size. [Background technology]

[0003] 3.Background Internalizing receptors are receptors that translocate from the plasma membrane to the interior of the cell when the cell surface receptor is monoubiquitinated after ligand-induced activation. These receptors are then taken up into endocytic vesicles, from which they are targeted to lysosomes or vacuoles for degradation or recycled back to the plasma membrane.

[0004] An example of an internalizing receptor is the mannose 6-phosphate receptor (M6PR), a transmembrane glycoprotein that targets enzymes to lysosomes. These receptors play an important role in transporting proteins from the Golgi complex and cell surface to lysosomes. Cation-independent (CI) M6PRs are multifunctional in that they bind two different classes of ligands on the cell surface: M6P-containing proteins and insulin-like growth factor 2 (IGF2). Therefore, CI-M6PRs are synonymously referred to as "M6PR" and "IGF2-R."

[0005] IGF2 plays an important role in metabolic regulation through three receptors: two tyrosine kinase receptors (IGF1 receptor and insulin receptor isoform A) and CI-M6PR. Stimulation of type 1 receptor tyrosine kinase induces a protein phosphorylation cascade that leads to biological effects, particularly insulin-mediated proliferation and increased expression of CI-M6PR, which mediates the endocytosis and clearance of IGF2. Lysosomal degradation of IGF2 is crucial because elevated IGF2 levels induce abnormal proliferation. Binding of CI-M6PR to IGF2 inhibits IGF2-stimulated DNA synthesis in hepatocytes and fibroblasts, and at physiological concentrations, this receptor can inhibit IGF2-mediated tumor growth.

[0006] Therefore, there is a need to develop internalizing receptor antigen-binding proteins (ABPs) that can be used in the treatment, diagnosis, and study of a variety of diseases, including cancer, lysosomal, and autoimmune diseases.

[0007] Additionally, targeting soluble or cell surface target molecules for internalization may be useful in the treatment of additional diseases.

[0008] For example, IgE, a soluble molecule, is known to interact with two major receptors, FcεRI and CD23 / FcεRII4, which are involved in different immunological processes. Allergen-specific IgE binds with high affinity to FcεRI expressed on immune effector cells such as basophils and mast cells. This interaction occurs via two asymmetric binding sites on the receptor and is stabilized by inducing a conformational change in IgE. Upon exposure to an allergen, cross-linking of IgE-bound FcεRI is induced, resulting in the immediate activation of allergic effector cells, ultimately leading to cellular degranulation and the release of vasoactive and proinflammatory mediators. The anti-IgE antibody omalizumab binds to free IgE and inhibits its binding to FcεRI on mast cells and basophils. Omalizumab has been approved for the treatment of severe persistent allergic asthma and chronic idiopathic urticaria. The therapeutic effects of anti-IgE have also been reported for allergic rhinitis, allergic bronchopulmonary aspergillosis, latex allergy, atopic dermatitis, allergic urticaria, and anaphylaxis.

[0009] In another example, the cell surface target molecule epidermal growth factor receptor (EGFR) regulates normal growth and differentiation, and dysregulation of this receptor, or one of the EGFR ligands, is involved in the development of many cancers. EGFR and insulin-like growth factor receptor (IFGR) play important roles in the tumorigenesis of several types of human cancers. Inhibition of receptor tyrosine kinases, such as EGFR, has emerged as an effective therapeutic strategy for certain human malignancies (for a review, see Roussidis AE, In Vivo. 2002 16(6):459-69). While targeted monotherapy can be initially effective in cancer treatment, treatment resistance often follows, likely as a result of upregulation of other signaling cascades (see, e.g., Nahta R et al., Breast Cancer Res. 2006 8(6):215 and Horn L et al., Clin Lung Cancer. 2007 8:S68-73). Matuzumab, a humanized immunoglobulin G(1) (IgG(1)) anti-EGFR monoclonal antibody, inhibits EGFR activation. Thus, there is a need to develop improved therapies involving targeting cell surface molecules such as EGFR. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Roussidis AE, In Vivo.2002 16(6):459-69 [Non-patent document 2] Nahta R et al.,Breast Cancer Res.2006 8(6):215 [Non-patent document 3] Horn L et al., Clin Lung Cancer.2007 8:S68-73 Summary of the Invention [Means for solving the problem]

[0011] 4. Overview Provided herein are novel antibody binding proteins (ABPs) that comprise a first antigen-binding moiety that has binding specificity for the internalization domain of the cation-independent mannose-6-phosphate receptor (CI-M6PR), and methods of using such ABPs. In some embodiments, the internalization domain is a fragment of human CI-M6PR.

[0012] Also provided herein are novel ABPs comprising a first binding moiety having binding specificity for human CI-M6PR and a target binding moiety having specificity for a soluble extracellular or cell surface target molecule, and methods of using such ABPs. In some embodiments, the target binding moiety is a second antigen binding moiety. In some embodiments, the target binding moiety is linked to the first antigen binding moiety, optionally via a linker.

[0013] Also provided herein is an ABP comprising a cargo moiety. In some embodiments, the cargo moiety is a polypeptide fused to either a first or second antigen-binding moiety. In some embodiments, the cargo moiety is linked to either the first or second antigen-binding moiety, optionally via a linker.

[0014] In some embodiments, the first antigen-binding portion specifically binds to a domain of human CI-M6PR selected from domain 1, domain 4, domain 5, domain 6, domain 7, and domain 8.

[0015] In some embodiments, the first antigen-binding moiety binds to human CI-M6PR with high affinity. In some embodiments, the first antigen-binding moiety binds to human CI-M6PR with a dissociation equilibrium constant (K D In some embodiments, the first antigen-binding moiety binds to CI-M6PR with a K of about 1 nM to about 500 nM. D In some embodiments, the first antigen-binding moiety binds to CI-M6PR with a K of about 10 nM to about 100 nM. DIn some embodiments, the first antigen-binding moiety binds to CI-M6PR with a K of about 100 nM to about 200 nM. D In some embodiments, the first antigen-binding moiety binds to CI-M6PR with a K of about 200 nM to about 300 nM. D In some embodiments, the first antigen-binding moiety binds to CI-M6PR with a K of about 300 nM to about 400 nM. D In some embodiments, the first antigen-binding moiety binds to CI-M6PR with a K of about 400 nM to about 500 nM. D It binds to CI-M6PR.

[0016] In some embodiments, the dissociation rate (K off ) is 1×10 -8 s -1 ~0.1s -1 In some embodiments, the K of the first antigen-binding moiety against CI-M6PR is off is 1 x 10 -6 s -1 ~1×10 -2 s -1 In some embodiments, the K of the first antigen-binding moiety against CI-M6PR is off is approximately 1 x 10 -5 s -1 The following is the result.

[0017] In some embodiments, the binding of the first antigen-binding moiety to human CI-M6PR is pH-dependent. In some embodiments, the first antigen-binding moiety is released from CI-M6PR at pH 7.4 or below. In some embodiments, the first antigen-binding moiety is released from CI-M6PR at pH 6.0 or below. In some embodiments, the first antigen-binding moiety is released from CI-M6PR at pH 5.5 or below. In some embodiments, the first antigen-binding moiety is released from CI-M6PR at pH 5.0 or below.

[0018] In some embodiments, the first antigen-binding portion comprises (a) a light chain CDR3 (LCDR3) having the sequence of SEQ ID NO: 76 and a heavy chain CDR3 (HCDR3) having the sequence of SEQ ID NO: 35, or (b) a light chain CDR3 having the sequence of SEQ ID NO: 78 and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 38, or (c) a light chain CDR3 having the sequence of SEQ ID NO: 81 and a heavy chain CDR3 having the sequence of SEQ ID NO: 41, or (d) a light chain CDR3 having the sequence of SEQ ID NO: 84 and a heavy chain CDR3 having the sequence of SEQ ID NO: 44, or (e) a light chain CDR3 having the sequence of SEQ ID NO: 85 and a heavy chain CDR3 having the sequence of SEQ ID NO: 46, or (f) a light chain CDR3 having the sequence of SEQ ID NO: 88 and a heavy chain CDR3 having the sequence of SEQ ID NO: 49, or (g) a light chain CDR3 having the sequence of SEQ ID NO: 91 and a heavy chain CDR3 having the sequence of SEQ ID NO: 52, or (h) a light chain CDR3 having the sequence of SEQ ID NO: 94 and a heavy chain CDR3 having the sequence of SEQ ID NO: 5 or (i) a light chain CDR3 having the sequence of SEQ ID NO: 96 and a heavy chain CDR3 having the sequence of SEQ ID NO: 55, or (j) a light chain CDR3 having the sequence of SEQ ID NO: 99 and a heavy chain CDR3 having the sequence of SEQ ID NO: 58, or (k) a light chain CDR3 having the sequence of SEQ ID NO: 101 and a heavy chain CDR3 having the sequence of SEQ ID NO: 60, or (l) a light chain CDR3 having the sequence of SEQ ID NO: 104 and a heavy chain CDR3 having the sequence of SEQ ID NO: 63, or (m) a light chain CDR3 having the sequence of SEQ ID NO: 107 and a heavy chain CDR3 having the sequence of SEQ ID NO: 66, or (n) a light chain CDR3 having the sequence of SEQ ID NO: 110 and a heavy chain CDR3 having the sequence of SEQ ID NO: 69, or (o) a light chain CDR3 having the sequence of SEQ ID NO: 113 and a heavy chain CDR3 having the sequence of SEQ ID NO: 72, or (p) a light chain CDR3 having the sequence of SEQ ID NO: 116 and a heavy chain CDR3 having the sequence of SEQ ID NO: 73.

[0019] In some embodiments, the first antigen-binding portion comprises (a) a light chain CDR1 (LCDR1) having the sequence of SEQ ID NO: 74 and a heavy chain CDR1 (HCDR1) having the sequence of SEQ ID NO: 33, and a light chain CDR2 (LCDR2) having the sequence of SEQ ID NO: 75 and a heavy chain CDR2 (HCDR2) having the sequence of SEQ ID NO: 34, or (b) a LCDR1 having the sequence of SEQ ID NO: 77 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 36, and a LCDR2 having the sequence of SEQ ID NO: 75 and a HCDR2 having the sequence of SEQ ID NO: 37, or (c) a light chain CDR2 (LCDR2) having the sequence of SEQ ID NO: 79 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 36, and a light chain CDR2 (LCDR2) having the sequence of SEQ ID NO: 75 and a heavy chain CDR2 (HCDR2) having the sequence of SEQ ID NO: 37, or (d) LCDR1 having the sequence of SEQ ID NO: 82 and heavy chain HCDR1 having the sequence of SEQ ID NO: 42, and LCDR2 having the sequence of SEQ ID NO: 83 and HCDR2 having the sequence of SEQ ID NO: 43, or (e) LCDR1 having the sequence of SEQ ID NO: 79 and heavy chain HCDR1 having the sequence of SEQ ID NO: 45, and LCDR2 having the sequence of SEQ ID NO: 80 and HCDR2 having the sequence of SEQ ID NO: 34. or (f) an LCDR1 having the sequence of SEQ ID NO: 86 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 47, and an LCDR2 having the sequence of SEQ ID NO: 87 and an HCDR2 having the sequence of SEQ ID NO: 48, or (g) an LCDR1 having the sequence of SEQ ID NO: 89 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 50, and an LCDR2 having the sequence of SEQ ID NO: 90 and an HCDR2 having the sequence of SEQ ID NO: 53, or (h) an LCDR1 having the sequence of SEQ ID NO: 92 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 50, and an LCDR2 having the sequence of SEQ ID NO: 93 2 and HCDR2 having the sequence of SEQ ID NO: 53, or (i) a heavy chain HCDR1 having the sequence of SEQ ID NO: 95 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 50, and a LCDR2 having the sequence of SEQ ID NO: 93 and a HCDR2 having the sequence of SEQ ID NO: 51, or (j) a heavy chain HCDR1 having the sequence of SEQ ID NO: 97 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 56, and a LCDR2 having the sequence of SEQ ID NO: 98 and a HCDR2 having the sequence of SEQ ID NO: 57, or (k) a heavy chain HCDR1 having the sequence of SEQ ID NO: 100 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 50,and an LCDR2 having the sequence of SEQ ID NO:93 and an HCDR2 having the sequence of SEQ ID NO:59, or (l) an LCDR1 having the sequence of SEQ ID NO:102 and a heavy chain HCDR1 having the sequence of SEQ ID NO:61, and an LCDR2 having the sequence of SEQ ID NO:103 and an HCDR2 having the sequence of SEQ ID NO:62, or (m) an LCDR1 having the sequence of SEQ ID NO:105 and a heavy chain HCDR1 having the sequence of SEQ ID NO:64, and an LCDR2 having the sequence of SEQ ID NO:106 and an HCDR2 having the sequence of SEQ ID NO:65, or (n) an LCDR1 having the sequence of SEQ ID NO:108 and and a heavy chain HCDR1 having the sequence of SEQ ID NO: 67, and an LCDR2 having the sequence of SEQ ID NO: 109 and an HCDR2 having the sequence of SEQ ID NO: 68, or (o) an LCDR1 having the sequence of SEQ ID NO: 111 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 70, and an LCDR2 having the sequence of SEQ ID NO: 112 and an HCDR2 having the sequence of SEQ ID NO: 71, or (p) an LCDR1 having the sequence of SEQ ID NO: 114 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 70, and an LCDR2 having the sequence of SEQ ID NO: 115 and an HCDR2 having the sequence of SEQ ID NO: 71.

[0020] In some embodiments, the first antigen-binding portion comprises a variable light chain (VL) having an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 17-32.

[0021] In some embodiments, the first antigen-binding portion comprises a variable heavy chain (VH) having an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 1-16.

[0022] In some embodiments, the first antigen-binding portion comprises a variable light chain (VL) having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 17-32.

[0023] In some embodiments, the first antigen-binding portion comprises a variable heavy chain (VH) having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 1-16.

[0024] In some embodiments, the first antigen-binding portion is an antibody fragment (eg, a single-chain variable fragment (scFv) or an antigen-binding fragment (Fab)).

[0025] In some embodiments, the ABPs disclosed herein are transported to lysosomes. In some embodiments, the ABPs are returned to the cell surface after internalization. In some embodiments, the ABPs are degraded intracellularly.

[0026] In some embodiments, the ABP comprises a multispecific (e.g., bispecific) antibody (see, e.g., Figure 1). In some embodiments, the multispecific antibody comprises a first antigen-binding portion and a second antigen-binding portion. In certain embodiments, the first antigen-binding portion is an antigen-binding fragment selected from a Fab, F(ab'), a single-chain antibody (e.g., scFv), a diabody, a triabody, a tetrabody, and a domain antibody. In some embodiments, the antibody is selected from a human antibody, a humanized antibody, or a chimeric antibody. In certain embodiments, the second antigen-binding portion is an antigen-binding fragment selected from a Fab, F(ab'), a single-chain antibody (e.g., scFv), a diabody, a triabody, a tetrabody, and a domain antibody. In some embodiments, the antibody is selected from a human antibody, a humanized antibody, or a chimeric antibody.

[0027] Also provided herein are methods for degrading soluble or cell-surface target molecules, the methods comprising: (a) contacting the target molecule with a multispecific antigen-binding protein (ABP), where the ABP comprises a first antigen-binding moiety that specifically binds to a cation-independent mannose-6-phosphate receptor (CI-M6PR) on the surface of a cell; and (b) transporting the ABP, target molecule, and CI-M6PR to a lysosome within the cell, where the target molecule is degraded within the lysosome. In some embodiments, the methods further comprise (c) transporting the ABP to the cell surface.

[0028] Also provided herein are methods for internalizing a target molecule, the methods comprising: (a) contacting the target molecule with an antigen binding protein (ABP) disclosed herein; and (b) internalizing the ABP into a cell. 5. Brief description of the drawings [Brief explanation of the drawings]

[0029] [Figure 1] FIG. 1 is a schematic diagram of a bispecific antigen-binding protein (ABP). [Figure 2A] Figure 2 shows a non-limiting example of a multispecific antigen-binding protein having a first antigen-binding moiety having a full-length antibody that targets M6PR and a second antigen-binding moiety having an scFv that binds to a target molecule such as IgE (Figure 2A) or EGFR (Figure 2B). [Figure 2B] Figure 2 shows a non-limiting example of a multispecific antigen-binding protein having a first antigen-binding moiety having a full-length antibody that targets M6PR and a second antigen-binding moiety having an scFv that binds to a target molecule such as IgE (Figure 2A) or EGFR (Figure 2B). [Figure 3A] Figure 3A shows a non-limiting example of a multispecific antigen-binding protein having a first antigen-binding moiety with an scFv that targets M6PR and a second antigen-binding moiety comprising a full-length antibody that binds to a target molecule such as IgE (Figure 3A) or EGFR (Figure 3B). [Figure 3B] Figure 3A shows a non-limiting example of a multispecific antigen-binding protein having a first antigen-binding moiety with an scFv that targets M6PR and a second antigen-binding moiety comprising a full-length antibody that binds to a target molecule such as IgE (Figure 3A) or EGFR (Figure 3B). [Figure 4-1] FIG. 1 shows cell surface and intracellular staining of human M6PR in WT and M6PR-KO K562 cells. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 5-1]Figure 1 shows mouse titer validation data using recombinant antigens and validated by flow-based assay using human cell lines (K562 WT, K562 M6PR-KO, Hela WT and Hela M6PR-KO) and cross-screened against two mouse cell lines (Dipak MC38 and 4T1). [Figure 5-2] Same as above. [Figure 6] FIG. 1 is a schematic diagram of biosensor preparation and binding events for affinity evaluation of the top 192 clones via Octet for binding / dissociation rates and KD to human M6PR. [Figure 7] Figure 1 provides baseline measurements for each biosensor loaded with a representative mIgG clone from the supernatant (and a poly-mIgG control). Additional biosensors were regenerated to measure additional analyte interactions (off-target binding to mouse M6PR-His6 and His). Binding curve data for representative clones are shown in the top panel. [Figure 8] FIG. 1 provides an overview of how monoclonal antibodies targeted to M6PR are screened for uptake activity. [Figure 9A] FIG. 1 shows response curves from binding assays of various anti-M6PR antibodies. [Figure 9B] FIG. 1 shows response curves from binding assays of various anti-M6PR antibodies. [Figure 9C] FIG. 1 shows response curves from binding assays of various anti-M6PR antibodies. [Figure 9D] FIG. 1 shows response curves from binding assays of various anti-M6PR antibodies. [Figure 9E] FIG. 1 shows response curves from binding assays of various anti-M6PR antibodies. [Figure 9F] FIG. 1 shows response curves from binding assays of various anti-M6PR antibodies. [Figure 9G] FIG. 1 shows response curves from binding assays of various anti-M6PR antibodies. [Figure 10]FIG. 1 shows a table of affinities (KD) and kinetics (Kon, Koff) for 15 anti-M6PR antibodies. [Figure 11] Schematic diagram showing an antibody that binds to domains D1-D9 (left panel) and D2 of human CI-M6PR. This antibody does not bind to chimeric CI-M6PR in which D2 of human CI-M6PR has been replaced with the corresponding domain from mouse CI-M6PR. Ligands for CI-M6PR are listed adjacent to the domains to which they bind. [Figure 12A] Diagram of the assay used for epitope binning. Coupled Ab is a probe-labeled anti-M6PR antibody. [Figure 12B] 1 shows binding curves, with each line representing a different bin based on the kinetics of the curve. [Figure 13-1] Figure 1 shows network analysis of epitope binning. The left panel shows the bin assignments for each of the top 12 anti-M6PR antibodies. The right panel shows the positions of bin 7, bin 17, and bin 3 within the overall network analysis. [Figure 13-2] Same as above. [Figure 14] FIG. 1 shows expression gels for each of the HIS-tagged D1 to D9 CI-M6PR domains. [Figure 15A] Figure 1 shows the binding curves of various anti-M6PR antibodies binding to mouse CI-M6PR D1-D9 domains (LT011-muD1-LT011muD9). Human wild-type CI-M6PR was used as a positive control (huLT011). [Figure 15B] Figure 1 shows the binding curves of various anti-M6PR antibodies binding to mouse CI-M6PR D1-D9 domains (LT011-muD1-LT011muD9). Human wild-type CI-M6PR was used as a positive control (huLT011). [Figure 15C] Figure 1 shows the binding curves of various anti-M6PR antibodies binding to mouse CI-M6PR D1-D9 domains (LT011-muD1-LT011muD9). Human wild-type CI-M6PR was used as a positive control (huLT011). [Figure 15D] Figure 1 shows the binding curves of various anti-M6PR antibodies binding to mouse CI-M6PR D1-D9 domains (LT011-muD1-LT011muD9). Human wild-type CI-M6PR was used as a positive control (huLT011). [Figure 15E] Figure 1 shows the binding curves of various anti-M6PR antibodies binding to mouse CI-M6PR D1-D9 domains (LT011-muD1-LT011muD9). Human wild-type CI-M6PR was used as a positive control (huLT011). [Figure 15F] Figure 1 shows the binding curves of various anti-M6PR antibodies binding to mouse CI-M6PR D1-D9 domains (LT011-muD1-LT011muD9). Human wild-type CI-M6PR was used as a positive control (huLT011). [Figure 15G] Figure 1 shows the binding curves of various anti-M6PR antibodies binding to mouse CI-M6PR D1-D9 domains (LT011-muD1-LT011muD9). Human wild-type CI-M6PR was used as a positive control (huLT011). [Figure 15H] Figure 1 shows the binding curves of various anti-M6PR antibodies binding to mouse CI-M6PR D1-D9 domains (LT011-muD1-LT011muD9). Human wild-type CI-M6PR was used as a positive control (huLT011). [Figure 16] FIG. 15B is a schematic diagram summarizing the domain mapping results of FIGS. 15A to 15H. [Figure 17A] FIG. 1 shows the experimental workflow for assessing pH association and dissociation of anti-M6PR antibodies from CI-M6PR. [Figure 17B] FIG. 1 shows the binding curves of clone 17F11 for association at pH 7.4 and dissociation at pH 5.0, pH 6.0, and pH 7.4. [Figure 17C] FIG. 1 shows the binding curves of clone 18G4 for association at pH 7.4 and dissociation at pH 5.0, pH 6.0, and pH 7.4. [Figure 17D]FIG. 1 shows the binding curves of clone 21D5 for association at pH 7.4 and dissociation at pH 5.0, pH 6.0, and pH 7.4. [Figure 18] Figure 1 shows a histogram quantifying the pH-dependent dissociation of nine different anti-M6PR antibody clones. pH-dependent dissociation was determined by dividing the Koff at pH 5.0 (pH 5.0 / 7.4) or pH 6.0 (pH 6.0 / pH 7.4) by the Koff at pH 7.4 for each antibody. Dissociation is quantified as the fold change in binding shift after 300 seconds at the indicated pH. [Figure 19A] FIG. 1 is a schematic diagram showing the steps and biology underlying the pH rodo uptake assay. [Figure 19B] Figure 1 shows a histogram of mean fluorescence intensity from the pH rodo uptake assay for 30 anti-M6PR antibodies. The dotted line represents the uptake of the KLH-IGF2 positive control. * indicates antibodies used at less than 25 nM due to low expression during production. [Figure 20A] Figure 1 shows the uptake curves of anti-M6PR-omalizumab bispecific antibodies. IgE uptake was measured by the mean fluorescence intensity (MFI) of Alexa Fluor 488 bound to IgE. Uptake was measured at various concentrations of bispecific antibody or antibody, as indicated on the x-axis. [Figure 20B] Figure 1 shows the uptake curves of anti-M6PR-omalizumab bispecific antibodies. IgE uptake was measured by the mean fluorescence intensity (MFI) of Alexa Fluor 488 bound to IgE. Uptake was measured at various concentrations of bispecific antibody or antibody, as indicated on the x-axis. [Figure 20C] Figure 1 shows the uptake curves of anti-M6PR antibodies. IgE uptake was measured by the mean fluorescence intensity (MFI) of Alexa Fluor 488 bound to IgE. Uptake was measured at various concentrations of bispecific antibody or antibody, as indicated on the x-axis. [Figure 20D]Figure 1 shows the uptake curves of anti-M6PR antibodies. IgE uptake was measured by the mean fluorescence intensity (MFI) of Alexa Fluor 488 bound to IgE. Uptake was measured at various concentrations of bispecific antibody or antibody, as indicated on the x-axis. [Figure 20E] Figure 1 shows the uptake curves of anti-M6PR antibodies. IgE uptake was measured by the mean fluorescence intensity (MFI) of Alexa Fluor 488 bound to IgE. Uptake was measured at various concentrations of bispecific antibody or antibody, as indicated on the x-axis. [Figure 20F] Figure 1 shows the uptake curves of anti-M6PR antibodies. IgE uptake was measured by the mean fluorescence intensity (MFI) of Alexa Fluor 488 bound to IgE. Uptake was measured at various concentrations of bispecific antibody or antibody, as indicated on the x-axis. [Figure 21] FIG. 1 provides a schematic diagram of the surface and total HiBit assays, as well as an exemplary readout for each assay. [Figure 22A] 1 is a histogram showing the span (ie, surface EGFR depletion) of 16 matuzumab-anti-M6PR bispecific antibodies. [Figure 22B] 1 is a histogram showing the span (ie, total EGFR depletion) of 16 matuzumab-anti-M6PR bispecific antibodies. [Figure 22C] FIG. 1 shows the percentage of surface EGFR activity of exemplary matuzumab-anti-M6PR bispecific antibodies (3C7, 53B11). [Figure 22D] FIG. 1 shows the percentage of total EGFR activity of exemplary matuzumab-anti-M6PR bispecific antibodies (3C7, 53B11). [Figure 23] FIG. 1 is a schematic diagram of the experimental workflow for the hIgE uptake assay using the omalizumab-anti-M6PR (MPR6-Oma) bispecific antibody. [Figure 24A] FIG. 1 shows internalization and recycling of M6PR6-Oma bispecific antibody by HepG2 cells (shown as mean fluorescence intensity (MFI)). [Figure 24B] Figure 24B shows quantification of internalization and recycling of the M6PR-Oma bispecific antibody shown in Figure 24A. The 0' condition was used as the internal baseline and set to 100%. Quantification was measured as hIgE surface labeling / uptake. [Figure 25A] FIG. 1 shows the internalization and recycling of MPR6-Oma bispecific antibodies by K562 cells (shown as mean fluorescence intensity (MFI)). [Figure 25B] Figure 25B shows quantification of internalization and recycling of the M6PR-Oma bispecific antibody shown in Figure 25A. The 0' condition was used as the internal baseline and set to 100%. Quantification was measured as hIgE surface labeling / uptake. [Figure 26A] FIG. 1 shows C5 uptake by anti-M6PR / anti-C5 bispecific antibody using C5 fluorescently labeled with pH rodo. [Figure 26B] FIG. 1 shows C5 uptake by anti-M6PR / anti-C5 bispecific antibody using C5 fluorescently labeled with AF647. [Figure 27A] Figure 1 shows that LUM-anti-M6PR bispecific antibodies mediated HER3 internalization and reduced cell viability in trastuzumab-resistant cells. Surface depletion (i.e., span) and total depletion (i.e., span) of HER3 for a subset of LUM-anti-M6PR bispecific antibodies are shown. Lumuletuzumab (LUM), a humanized anti-human epidermal growth factor receptor 3 (HER3) monoclonal antibody, and NRG1 (neuregulin 1) antibody were used as positive controls. A non-binding antibody (KLH) fused to LUM was used as a negative control for uptake activity. [Figure 27B] Figure 1 shows that LUM-anti-M6PR bispecific antibodies mediated HER3 internalization and reduced cell viability of trastuzumab-resistant cells. Cell viability after treatment with a subset of LUM-anti-M6PR bispecific antibodies is shown. DETAILED DESCRIPTION OF THE INVENTION

[0030] 6. Detailed Description 6.1.Definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art. Unless otherwise specified, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and described throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. The terminology used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery, and treatment of patients.

[0031] Unless otherwise stated, the following terms shall be understood to have the following meanings:

[0032] The terms "M6PR," "M6PR protein," and "M6PR antigen" are used interchangeably herein and refer to human M6PR, human CI-M6PR, or any variants (e.g., splice and allelic variants), isoforms, and species homologs of human M6PR naturally expressed by cells or expressed by cells transfected with the M6PR gene. In some embodiments, the M6PR protein is an M6PR protein naturally expressed by a primate (e.g., monkey or human), rodent (e.g., mouse or rat), dog, camel, cat, cow, goat, horse, or sheep. In some embodiments, the M6PR protein is human cation-independent M6PR (NCBI Accession No. NP_000867, SEQ ID NO: 276).

[0033] The term "internalizing receptor" refers to a receptor that can move from an external region (including, for example, the cell surface) to the interior of a cell. Internalizing receptors include, but are not limited to, endocytic receptors and receptors that transport to lysosomes. Non-limiting examples of internalizing receptors include mannose-6-phosphate receptor (CD-M6PR) and cation-independent mannose-6-phosphate receptor (CI-M6PR).

[0034] The term "immunoglobulin" generally refers to a class of structurally related proteins that contain two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In "intact immunoglobulins," all four chains are interconnected by disulfide bonds. The structure of immunoglobulins is well-characterized. See, e.g., Paul, Fundamental Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy chain typically contains a heavy chain variable region (V H) and heavy chain constant region (C H The heavy chain constant region typically contains C H1 , C H2 , and C H3 Each light chain typically contains three domains, abbreviated as V L ) and a light chain constant region. The light chain constant region is usually C L It contains one domain, abbreviated as .

[0035] The term "antigen binding protein" (ABP) refers to a protein comprising one or more antigen binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen binding domain binds to an antigen or epitope with specificity and affinity similar to a naturally occurring antibody. In some embodiments, the ABP comprises an antibody. In some embodiments, the ABP consists essentially of an antibody. In some embodiments, the ABP comprises an alternative scaffold. In some embodiments, the ABP consists essentially of an alternative scaffold. In some embodiments, the ABP comprises an antibody fragment. In some embodiments, the ABP consists essentially of an antibody fragment. In some embodiments, the ABP consists essentially of an antibody fragment. An "internalizing receptor ABP," "anti-internalizing receptor ABP," or "internalizing receptor-specific ABP," as provided herein, is an ABP that specifically binds to the antigen of an internalizing receptor. In some embodiments, the ABP binds to the extracellular domain of an internalizing receptor. In certain embodiments, the internalizing receptor ABPs provided herein bind to an epitope of an internalizing receptor that is conserved between or among internalizing receptor proteins from different species. A "soluble target molecule ABP," an "anti-soluble target molecule ABP," or a "soluble target molecule-specific ABP," as provided herein, is an ABP that specifically binds to an antigen of a soluble target molecule. In some embodiments, the ABP binds to the extracellular domain of a soluble target molecule. In certain embodiments, the soluble target molecule ABP provided herein binds to an epitope of a soluble target molecule that is conserved between or among soluble target molecule proteins from different species. A "cell surface target molecule ABP," an "anti-cell surface target molecule ABP," or a "cell surface target molecule-specific ABP," as provided herein, is an ABP that specifically binds to an antigen of a cell surface target molecule. In some embodiments, the ABP binds to the extracellular domain of a cell surface target molecule. In certain embodiments, the cell surface target molecule ABP provided herein binds to an epitope of a cell surface target molecule that is conserved between or among cell surface target molecule proteins from different species.

[0036] The term "antibody" is used herein in its broadest sense and includes a specific type of immunoglobulin molecule that contains one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies. An example of an antigen-binding domain is a V H -V L An antigen-binding domain formed by a dimer. An antibody is a type of ABP.

[0037] The term "bispecific ABP" or "multispecific" refers to an ABP (e.g., an antibody) that has at least two different antigen-binding moieties (e.g., antigen-binding domains), each of which specifically binds to a different antigen, e.g., an internalizing receptor and a soluble or cell-surface target molecule. Bispecific ABPs can be of any number of valencies, e.g., bivalent, trivalent, tetravalent, etc.

[0038] The term "multispecific ABP" refers to an ABP (e.g., an antibody) having at least two (e.g., at least three, at least four, at least five, at least six, etc.) different antigen-binding moieties (e.g., antigen-binding domains), each of which specifically binds to a different antigen, e.g., an internalizing receptor and a soluble or cell-surface target molecule. A bispecific ABP can be of any number of valencies, e.g., bivalent, trivalent, tetravalent, etc. The term "antigen-binding moiety" or "antigen-binding domain" refers to a portion of an ABP that can specifically bind to an antigen or epitope.

[0039] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a naturally occurring antibody structure and having a heavy chain that includes an Fc region.

[0040] The term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain that, in naturally occurring antibodies, interacts with Fc receptors and specific proteins of the complement system. The structures of the Fc regions of various immunoglobulins and the glycosylation sites contained therein are well known in the art. See Schroeder and Cavacini, J. Allergy Clin. Immunol., 2010, 125:S41-52 (incorporated by reference in its entirety). The Fc region may be a naturally occurring Fc region or an altered Fc region as described elsewhere in this disclosure.

[0041] V H Area and V L The regions can be further subdivided into regions of hypervariability (also called "hypervariable regions (HVRs)" or "complementarity determining regions (CDRs)") interspersed with more conserved regions. The more conserved regions are called framework regions (FRs). Each V H and V L generally contain three CDRs and four FRs arranged in the following order (from N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved in antigen binding and affect the antigen specificity and binding affinity of the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, MD (incorporated by reference in its entirety).

[0042] Light chains from any vertebrate species can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the sequence of their constant domain.

[0043] Heavy chains from all vertebrate species can be classified into one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also called α, δ, ε, γ, and μ, respectively. The IgG and IgA classes are further divided into subclasses based on sequence and functional differences. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0044] The amino acid sequence boundaries of the CDRs can be determined by one of skill in the art using any of several known numbering schemes, including those described by Kabat et al., supra (the "Kabat" numbering scheme), Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the "Chothia" numbering scheme), MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (the "Contact" numbering scheme), Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (the "IMGT" numbering scheme), and Honegge and Plueckthun, J. Mol. Biol., 2001, 309:657-70 (the "AHo" numbering scheme), each of which is incorporated by reference in its entirety.

[0045] Table 1 shows the CDR1-L(V) sequences identified by the Kabat and Chothia scheme. L CDR1), CDR2-L(V L CDR2), CDR3-L(V L CDR3), CDR1-H(V H CDR1), CDR2-H(V H CDR2 of V), and CDR3-H (V H For CDR1-H, residue numbering is provided using both the Kabat and Chothia numbering schemes.

[0046] CDRs can be assigned using antibody numbering software such as Abnum, available at www.bioinf.org.uk / abs / abnum / and described in Abhinandan and Martin, Immunology, 2008, 45:3832-3839 (incorporated by reference in its entirety). [Table 1] *The C-terminus of CDR1-H, numbered using the Kabat numbering convention, varies between 32 and 34 depending on the length of the CDR.

[0047] The "EU numbering scheme" is commonly used when referring to residues within antibody heavy chain constant regions (eg, as reported in Kabat et al., supra).

[0048] "Antibody fragments" include portions of intact antibodies, such as the antigen-binding or variable regions of the intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0049] An "Fv" fragment comprises a non-covalent dimer of one heavy- and one light-chain variable domain.

[0050] A "Fab" fragment contains the heavy and light chain variable domains as well as the constant domain of the light chain and the first constant domain of the heavy chain (C H1 ) Fab fragments can be produced, for example, by recombinant methods or by papain digestion of a full-length antibody.

[0051] An "F(ab')2" fragment contains two Fab' fragments linked by a disulfide bond near the hinge region. F(ab')2 fragments can be produced, for example, by recombinant methods or by pepsin digestion of an intact antibody. F(ab')2 fragments can be dissociated, for example, by treatment with β-mercaptoethanol.

[0052] "Single-chain Fv" or "sFv" or "scFv" antibody fragments contain V in a single polypeptide chain. H Domain and V L Includes domain. V H and V L are generally linked by a peptide linker. See Plueckthun A. (1994). In some embodiments, the linker is (GGGGS) n In some embodiments, n=1, 2, 3, 4, 5, or 6. See Antibodies from Escherichia coli. In Rosenberg M. & Moore GP (Eds.), The Pharmacology of Monoclonal Antibodies vol. 113 (pp. 269-315). Springer-Verlag, New York (incorporated by reference in its entirety).

[0053] An "scFv-Fc" fragment comprises an scFv linked to an Fc domain. For example, the Fc domain may be linked to the C-terminus of the scFv. The ..., depending on the orientation of the variable domains within the scFv (i.e., V H -V L or V L -V H ) depending on V H or V L Any suitable Fc domain known in the art or described herein can be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[0054] The term "single domain antibody" refers to a molecule in which one variable domain of an antibody specifically binds to an antigen in the absence of other variable domains. Single domain antibodies and fragments thereof are described in Arabi Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans et al., Trends in Biochem. Sci., 2001, 26:230-245, each of which is incorporated by reference in its entirety.

[0055] A "monospecific ABP" is an ABP that contains a binding site that specifically binds to a single epitope. An example of a monospecific ABP is a naturally occurring IgG molecule, which is bivalent but recognizes the same epitope in each antigen-binding domain. The binding specificity may exist in any suitable valency.

[0056] The term "monoclonal antibody" refers to an antibody from a substantially homogeneous population of antibodies. A substantially homogeneous population of antibodies contains antibodies that are substantially similar and bind to the same epitope(s), except for variants that may normally arise during the production of monoclonal antibodies. Such variants are generally present in small amounts. Monoclonal antibodies are typically obtained by a process of selecting a single antibody from multiple clones. For example, the selection process can be the selection of a unique clone from multiple clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further modified, for example, to improve its affinity for the target ("affinity maturation"), to humanize the antibody, to improve its production in cell culture, and / or to reduce its immunogenicity in a subject.

[0057] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remainder of the heavy and / or light chain is derived from a different source or species.

[0058] "Humanized" forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Humanized antibodies are generally human antibodies (recipient antibodies) in which residues from one or more CDRs have been replaced with residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody (such as a mouse, rat, rabbit, chicken, or non-human primate antibody) that has the desired specificity, affinity, or biological effect. In some cases, selected framework region residues of the recipient antibody are replaced with corresponding framework region residues from the donor antibody. Humanized antibodies can also contain residues that are not found in either the recipient antibody or the donor antibody. Such modifications can be made to further refine antibody function. For further details, see Jones et al., Nature, 1986, 321:522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596 (each of which is incorporated by reference in its entirety).

[0059] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a non-human source that utilizes the human antibody repertoire or human antibody coding sequences (e.g., obtained from a human source or designed de novo). Human antibodies specifically do not include humanized antibodies.

[0060] An "isolated ABP" or "isolated nucleic acid" is an ABP or nucleic acid that has been separated and / or recovered from a component of its natural environment. Components of natural environment may include enzymes, hormones, and other proteinaceous or non-proteinaceous substances. In some embodiments, an isolated ABP is purified sufficiently to obtain at least 15 residues of N-terminal or internal amino acid sequence, for example, by using a spinning cup sequencer. In some embodiments, an isolated ABP is purified to homogeneity by gel electrophoresis (e.g., SDS-PAGE) under reducing or non-reducing conditions and detected by Coomassie blue or silver staining. Isolated ABPs include ABPs in situ within recombinant cells, since at least one component of the ABP's natural environment is absent. In some embodiments, an isolated ABP or isolated nucleic acid is prepared by at least one purification step. In some embodiments, an isolated ABP or isolated nucleic acid is purified to at least 80%, 85%, 90%, 95%, or 99% by weight. In some embodiments, the isolated ABP or isolated nucleic acid is purified to at least 80%, 85%, 90%, 95%, or 99% by volume. In some embodiments, the isolated ABP or isolated nucleic acid is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99%, or 100% by volume of the ABP or nucleic acid. In some embodiments, the isolated ABP or isolated nucleic acid is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99%, or 100% by volume of the ABP or nucleic acid.

[0061] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an ABP) and its binding partner (e.g., an antigen or epitope). Unless otherwise indicated, as used herein, "affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an ABP and an antigen or epitope). The affinity of a molecule X for its partner Y is determined by the dissociation equilibrium constant (K D) The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

[0062] With respect to the binding of an ABP to a target molecule, the terms "bind," "specific binding," "specifically binds," "specific," "selectively binds," and "selective" to a particular antigen (e.g., a polypeptide target) or epitope on a particular antigen refer to binding that is distinct from nonspecific or nonselective interactions (e.g., interactions with non-target molecules). Specific binding can be measured, for example, by measuring binding to the target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In this case, specific binding is indicated if binding of the ABP to the target molecule is competitively inhibited by the control molecule. For example, in some embodiments, the affinity of the internalizing receptor ABP for the non-target molecule is less than about 50% of the affinity for the internalizing receptor. In some embodiments, the affinity of the internalizing receptor ABP for the non-target molecule is less than about 40% of the affinity for the internalizing receptor. In some embodiments, the affinity of the internalizing receptor ABP for the non-target molecule is less than about 30% of the affinity for the internalizing receptor. In some embodiments, the affinity of the internalizing receptor ABP for the non-target molecule is less than about 20% of its affinity for the internalizing receptor. In some embodiments, the affinity of the internalizing receptor ABP for the non-target molecule is less than about 10% of its affinity for the internalizing receptor. In some embodiments, the affinity of the internalizing receptor ABP for the non-target molecule is less than about 1% of its affinity for the internalizing receptor. In some embodiments, the affinity of the internalizing receptor ABP for the non-target molecule is less than about 0.1% of its affinity for the internalizing receptor.

[0063] The term "k" d " (sec -1 ) as used herein refers to the dissociation rate constant of a particular ABP-antigen interaction. This value is known as the K off Also called value.

[0064] The term "k" a " (M -1 ×sec -1 ) as used herein refers to the association rate constant of a particular ABP-antigen interaction. This value is known as the K on Also called value.

[0065] The term “K D " (M), as used herein, refers to the dissociation equilibrium constant of a particular ABP-antigen interaction. K D =k d / k a .

[0066] The term “K A " (M -1 ) as used herein refers to the association equilibrium constant of a particular ABP-antigen interaction. K A =k a / k d .

[0067] An "affinity matured" ABP is an ABP with one or more modifications (e.g., in one or more CDRs or FRs) that result in an improvement in the affinity of the ABP for its antigen compared to a parent ABP that does not have the modification(s). In one embodiment, the affinity matured ABP has nanomolar or picomolar affinity for the target antigen. Affinity matured ABPs can be generated using a variety of methods well known in the art. For example, Marks et al. (Bio / Technology, 1992, 10:779-783, incorporated by reference in its entirety) describe the affinity matured ABP V. H and V LAffinity maturation by domain shuffling is described. Random mutagenesis of CDR and / or framework residues is described, for example, in Barbas et al. (Proc. Nat. Acad. Sci. USA, 1994, 91:3809-3813), Schier et al., Gene, 1995, 169:147-155, Yelton et al., J. Immunol., 1995, 155:1994-2004, Jackson et al., J. Immunol., 1995, 154:3310-33199, and Hawkins et al., J. Mol. Biol., 1992, 226:889-896, each of which is incorporated by reference in its entirety.

[0068] An "immunoconjugate" is an ABP bound to one or more heterologous molecule(s).

[0069] "Effector function" refers to a biological activity mediated by the Fc region of an antibody, which may vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding to activate complement-dependent cytotoxicity (CDC), Fc receptor binding to activate antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).

[0070] As used herein in the context of two or more ABPs, the terms "compete with" or "cross-compete with" refer to two or more ABPs competing for binding to an antigen (e.g., an internalizing receptor). In one exemplary assay, an internalizing receptor is coated on a surface and contacted with a first internalizing receptor ABP, followed by the addition of a second internalizing receptor ABP. In another exemplary assay, a first internalizing receptor is coated on a surface and contacted with an internalizing receptor, followed by the addition of a second internalizing receptor ABP. ABPs compete if the presence of the first internalizing receptor ABP reduces the binding of the second internalizing receptor ABP in either assay. The term "compete with" also includes combinations of ABPs in which one ABP reduces the binding of another ABP, but no competition is observed when the ABPs are added in the reverse order. However, in some embodiments, the first and second ABPs inhibit each other's binding, regardless of the order in which they are added. In some embodiments, one ABP reduces the binding of another ABP to its antigen by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%. One skilled in the art can select the concentration of antibody to use in a competition assay based on the affinity of the ABP for the internalizing receptor and the valency of the ABP. The assays described in this definition are exemplary, and one skilled in the art can utilize any suitable assay to determine whether antibodies compete with each other. Suitable assays are described, for example, in Cox et al., "Immunoassay Methods," in Assay Guidance Manual [Internet], updated December 24, 2014 (www.ncbi.nlm.nih.gov / books / NBK92434 / , accessed September 29, 2015), Silman et al., Cytometry, 2001, 44:30-37, and Finco et al., J. Pharm. Biomed. Anal., 2011, 54:351-358, each of which is incorporated by reference in its entirety.

[0071] The term "epitope" refers to a portion of an antigen that specifically binds to an ABP. Epitopes often consist of surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural and charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former may be lost in the presence of denaturing solvents, but the binding to the latter is not lost. An epitope may include amino acid residues directly involved in binding as well as other amino acid residues not directly involved in binding. The epitope to which an ABP binds can be determined using known techniques for determining epitopes, such as testing the binding of an ABP to internalizing receptors, soluble target molecules, and / or cell surface target molecule mutants with different point mutations, or chimeric internalizing receptors, soluble target molecules, and / or cell surface target molecule mutants.

[0072] The " identity " percentage between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage.The alignment for determining the amino acid sequence identity percentage can be achieved in various ways within the skill of the art, for example, by using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA or MUSCLE software.Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences to be compared.

[0073] "Conservative substitution" or "conservative amino acid substitution" refers to the replacement of an amino acid with a chemically or functionally similar amino acid. Conservative substitution tables providing similar amino acids are well known in the art. For example, the groups of amino acids provided in Tables 2-4 are, in some embodiments, considered conservative substitutions for each other. [Table 2] [Table 3] [Table 4]

[0074] Further conservative substitutions can be found, for example, in Creighton, Proteins: Structures and Molecular Properties 2nd ed. (1993) W.H. Freeman & Co., New York, NY. ABPs generated by making one or more conservative substitutions of amino acid residues in a parent ABP are referred to as "conservatively modified variants."

[0075] The term "treating" (and variations thereof, such as "treat" or "treatment") refers to a clinical intervention that seeks to alter the natural course of a disease or condition in a subject in need of treatment. Treatment can be prophylactic or can occur during the course of clinical pathology. Desirable effects of treatment include prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, improvement or palliation of the disease state, and remission or improved prognosis.

[0076] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of an ABP or pharmaceutical composition provided herein that, when administered to a subject, is effective to treat a disease or disorder.

[0077] As used herein, the term "subject" refers to a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject suffers from a disease or condition that can be treated with the ABPs provided herein. In some aspects, the disease or condition is cancer. In some aspects, the disease or condition is a viral infection.

[0078] The term "package insert" refers to instructions typically included in the commercial packaging of a therapeutic or diagnostic product (e.g., a kit) that contain information regarding the indications, methods of use, dosage, administration, concomitant therapy, contraindications, and / or warnings for the use of such therapeutic or diagnostic product.

[0079] The term "cytotoxic agent," as used herein, refers to a substance that inhibits or prevents the function of cells and / or causes the death or destruction of cells.

[0080] "Chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Chemotherapeutic agents include "antihormonal agents" or "endocrine therapeutic agents" that act to regulate, reduce, block, or inhibit the effects of hormones that can promote cancer growth.

[0081] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein. The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is cancer.

[0082] The term "pharmaceutical composition" refers to a preparation in which the biological activity of the active ingredient contained therein is in a form such that it is effective in treating a subject, and which does not contain additional ingredients that are unacceptably toxic to the subject.

[0083] The terms "modulate" and "modulation" refer to decreasing or inhibiting, or activating or increasing, the recited variable.

[0084] The terms "increase" and "activate" refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater increase in the recited variable.

[0085] The terms "reduce" and "inhibit" refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater decrease in the recited variable.

[0086] The term "agonize" refers to activating receptor signaling to induce a biological response associated with receptor activation. An "agonist" is an entity that binds to and agonizes a receptor.

[0087] The term "antagonize" refers to inhibiting receptor signaling to inhibit the biological response associated with receptor activation. An "antagonist" is an entity that binds to and antagonizes a receptor.

[0088] A "variant" of a polypeptide (e.g., an antibody) includes an amino acid sequence in which one or more amino acid residues have been inserted into, deleted from, and / or substituted into the amino acid sequence compared to the native polypeptide sequence, and which retains essentially the same biological activity as the native polypeptide. The biological activity of a polypeptide can be measured using standard techniques in the art (e.g., if the variant is an antibody, its activity can be tested by binding assays as described herein). Variants of the present invention include fragments, analogs, recombinant polypeptides, synthetic polypeptides, and / or fusion proteins.

[0089] A "derivative" of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, for example, by conjugation to another chemical moiety, such as polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation. Unless otherwise indicated, the term "antibody" includes antibodies comprising two full-length heavy chains and two full-length light chains, as well as derivatives, mutants, fragments, and muteins thereof, examples of which are described below.

[0090] A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., level, timing, or location of expression) of the nucleotide sequence. A "regulatory sequence" is a nucleic acid that affects the expression (e.g., level, timing, or location of expression) of a nucleic acid to which it is operably linked. A regulatory sequence can exert its effect, for example, directly on the nucleic acid being regulated or through the action of one or more other molecules (e.g., a polypeptide that binds to the regulatory sequence and / or nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Further examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA, and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.

[0091] A "host cell" is a cell that can be used to express a nucleic acid, e.g., a nucleic acid of the invention. A host cell can be a prokaryote, e.g., E. coli, or a eukaryote, e.g., a unicellular eukaryote (e.g., yeast or other fungus), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell), or a hybridoma. Examples of host cells include CS-9 cells, the monkey kidney cell line COS-7 (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivatives (such as Veggie CHO) and related cell lines that grow in serum-free medium (see Rasmussen et al., 1998, Cytotechnology 28:31), HeLa cells, the BHK (ATCC CRL 10) cell line, the CV1 / EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) (see McMahan et al., 1991, EMBO J. 10:2821), 293, 293 EBNA, or MSR cells. Examples of host cells include human embryonic kidney cells such as 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK, or Jurkat cells. Typically, host cells are cultured cells that can be transformed or transfected with a nucleic acid encoding a polypeptide, which can then be expressed in the host cell.

[0092] The phrase "recombinant host cell" can be used to refer to a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell may also be a cell that contains a nucleic acid but does not express the nucleic acid at a desired level unless a regulatory sequence is introduced into the host cell so that the host cell is operably linked to the nucleic acid. It is understood that the term host cell refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in subsequent generations due to, for example, mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but still be within the scope of the term as used herein.

[0093] 6.2. Other Interpretation Rules Ranges recited herein are understood to be shorthand for all values within the range, inclusive of the recited endpoints. For example, the range 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0094] Unless otherwise stated, a reference to a compound having one or more stereocenters contemplates each and every stereoisomer and all combinations of stereoisomers thereof.

[0095] 6.3. Nucleic acids In one aspect, the present invention provides isolated nucleic acid molecules. Nucleic acids include, for example, polynucleotides encoding all or a portion of an antigen-binding protein, e.g., one or both strands of an antibody of the invention, or a fragment, derivative, mutein, or variant thereof; polynucleotides sufficient for use as hybridization probes; PCR or sequencing primers for identifying, analyzing, mutating, or amplifying polynucleotides encoding polypeptides; antisense nucleic acids for inhibiting expression of polynucleotides; and complementary sequences to the foregoing. Nucleic acids can be of any length. Nucleic acids can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000, or more nucleotides in length, and / or can include one or more additional sequences, e.g., regulatory sequences, and / or can be part of a larger nucleic acid, e.g., a vector. Nucleic acids may be single-stranded or double-stranded and may comprise RNA and / or DNA nucleotides, as well as artificial variants thereof (eg, peptide nucleic acids).

[0096] Nucleic acids encoding antibody polypeptides (e.g., heavy or light chains, variable domains only, or full-length) can be isolated from cells of mice immunized with an internalizing receptor, a soluble target molecule, and / or a cell surface target molecule. Nucleic acids can be isolated by conventional procedures, such as polymerase chain reaction (PCR).

[0097] Nucleic acid sequences encoding the heavy chain variable region and the light chain variable region are provided herein. Those skilled in the art will understand that due to the degeneracy of the genetic code, each of the polypeptide sequences disclosed herein is encoded by numerous other nucleic acid sequences. The present invention provides each degenerate nucleotide sequence encoding each antigen-binding protein of the present invention.

[0098] The present invention further provides nucleic acids that hybridize to other nucleic acids (e.g., nucleic acids containing the nucleotide sequence of any of internalization receptor, soluble target molecule, and / or cell surface target molecule genes) under specific hybridization conditions. Methods for hybridizing nucleic acids are well known in the art. See, for example, Curr. Prot. in Mol. Biol., John Wiley & Sons, NY (1989), 6.3.1-6.3.6. As defined herein, moderately stringent hybridization conditions use a hybridization buffer of 5x sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), approximately 50% formamide, a pre-wash solution containing 6x SSC with a hybridization temperature of 55°C (or other similar hybridization solutions, such as those containing approximately 50% formamide with a hybridization temperature of 42°C), and wash conditions in 0.5x SSC, 0.1% SDS at 60°C. Stringent hybridization conditions involve hybridization in 6x SSC at 45°C, followed by one or more washes in 0.1x SSC, 0.2% SDS at 68°C. Moreover, one skilled in the art can manipulate hybridization and / or wash conditions to increase or decrease the stringency of hybridization, such that nucleic acids containing nucleotide sequences that are at least 65, 70, 75, 80, 85, 90, 95, 98, or 99% identical to each other will typically remain hybridized to each other.Basic parameters influencing the selection of hybridization conditions and guidance for devising appropriate conditions are described, for example, in Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11, and Curr. Prot. in Mol. Biol. 1995, Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4), and can be readily determined by one of skill in the art based, for example, on the length and / or base composition of the DNA.

[0099] Changes can be introduced into nucleic acids by mutation, thereby resulting in changes in the amino acid sequence of a polypeptide (e.g., an antigen-binding protein) that the nucleic acid encodes. Mutations can be introduced using any technique known in the art. In one embodiment, one or more specific amino acid residues are altered, for example, using a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are altered, for example, using a random mutagenesis protocol. However generated, the mutant polypeptides can be expressed and screened for desired properties (e.g., binding to an internalizing receptor, a soluble target molecule, and / or a cell surface target molecule).

[0100] 6.4. Antibodies Internalizing receptor antibodies can be purified from host cells transfected with antibody-encoding genes by eluting the filtered host cell culture supernatant using a heparin HP column with a salt gradient.

[0101] Fab fragments are V L , V H , C L , and C H1The F(ab')2 fragment is a monovalent fragment having two Fab fragments linked by a disulfide bond at the hinge region, and the Fd fragment is a V H Domain and C H1 The Fv fragment has a V domain, and the Fv fragment has a V domain of a single arm of the antibody. L and V H domain, and the dAb fragment has a V H Domain, V L Domain, or V H Or V L and antigen-binding fragments of the domain (U.S. Patent Nos. 6,846,634, 6,696,245, U.S. Patent Application Publication Nos. 05 / 0202512, 04 / 0202995, 04 / 0038291, 04 / 0009507, 03 / 0039958, Ward et al., Nature 341:544-546, 1989).

[0102] The polynucleotide and polypeptide sequences of particular light and heavy chain variable domains are set forth below. Antibodies comprising light and heavy chains are named by combining the name of the light chain variable domain with the name of the heavy chain variable domain. In some embodiments, the light chain variable sequences are provided in SEQ ID NOS: 32-63 and the heavy chain variable sequences are provided in SEQ ID NOS: 1-31.

[0103] In other embodiments, an antibody may comprise a particular heavy or light chain, but the complementary light or heavy chain variable domain remains unspecified. In particular, certain embodiments herein include antibodies that bind to a particular antigen (such as M6PR) via a particular light or heavy chain, such that the complementary heavy or light chain may be promiscuous or even unrelated, but can be determined, for example, by screening a combinatorial library. Portolano et al., J.Immunol.V.150(3), pp.880-887(1993), Clackson et al., Nature v.352 pp.624-628(1991), Adler et al., A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library, MAbs(2018), Adler et al. al., Rare, high-affinity mouse anti-PD-1 antibodies that function in checkpoint blockade, discovered using microfluidics and molecular genomics, MAbs (2017).

[0104] Naturally occurring immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) linked by three hypervariable regions, also called complementarity-determining regions or CDRs. From the N-terminus to the C-terminus, both light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain follows the definition in Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991.

[0105] The term "human antibody," also known as a "fully human antibody," includes all antibodies having one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (fully human antibodies). These antibodies can be prepared in a variety of ways, examples of which are described below, including immunizing with the antigen of interest mice that have been genetically engineered to express antibodies derived from genes encoding human heavy and / or light chains.

[0106] A humanized antibody has a sequence that differs from that of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and / or additions, such that the humanized antibody is less likely to induce an immune response and / or induces a milder immune response when administered to a human subject compared to an antibody from the non-human species. In one embodiment, specific amino acids within the framework and constant domains of the heavy and / or light chains of a non-human species antibody are mutated to generate a humanized antibody. In another embodiment, the constant domain(s) of a human antibody are fused to the variable domain(s) of a non-human species. In another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are altered to reduce the potential immunogenicity of the non-human antibody when administered to a human subject, where the altered amino acid residue is not important for immunospecific binding of the antibody to its antigen or the changes made to the amino acid sequence are conservative changes such that binding of the humanized antibody to the antigen is not significantly worse than binding of the non-human antibody to the antigen. Examples of how to make humanized antibodies can be found in US Pat. Nos. 6,054,297, 5,886,152, and 5,877,293.

[0107] The term "chimeric antibody" refers to an antibody that comprises one or more regions from one antibody and one or more regions from one or more other antibodies. In one embodiment, one or more of the CDRs are derived from a human anti-internalizing receptor antibody. In another embodiment, all of the CDRs are derived from a human anti-internalizing receptor antibody. In another embodiment, CDRs from multiple human anti-internalizing receptor antibodies are mixed and matched in a chimeric antibody. For example, a chimeric antibody can comprise CDR1 from the light chain of a first human anti-internalizing receptor antibody, CDR2 and CDR3 from the light chain of a second human anti-internalizing receptor antibody, and CDRs from the heavy chain of a third anti-internalizing receptor antibody. Furthermore, the framework regions can be derived from one of the same anti-internalizing receptor antibodies, one or more different antibodies (such as a human antibody), or a humanized antibody. In one example of a chimeric antibody, a portion of the heavy and / or light chain is identical to, homologous to, or derived from an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to, homologous to, or derived from an antibody(ies) from another species or belonging to another antibody class or subclass. Also included are fragments of such antibodies that exhibit the desired biological activity (i.e., the ability to specifically bind to an internalizing receptor).

[0108] Antibody fragments or analogs can be readily prepared by those skilled in the art using techniques well known in the art following the teachings of this specification. Preferred amino and carboxy termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data to public or proprietary sequence databases. Computerized comparison methods can be used to identify sequence motifs or predicted protein conformation domains present in other proteins of known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known. See, e.g., Bowie et al., 1991, Science 253:164.

[0109] Antigen-binding fragments derived from antibodies can be obtained, for example, by proteolytic hydrolysis of antibodies, e.g., pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be generated by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment called F(ab')2. This fragment can be further cleaved using a thiol reducing agent to generate a 3.5S Fab' monovalent fragment. If necessary, the cleavage reaction can be carried out using a blocking group for the sulfhydryl groups resulting from cleavage of disulfide bonds. Alternatively, enzymatic cleavage using papain directly generates two monovalent Fab fragments and one Fc fragment. These methods are described, for example, in Goldenberg, U.S. Pat. No. 4,331,647; Nisonoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., in Methods in Enzymology 1:422 (Academic Press 1967); and by Andrews, S. M. and Titus, J. A. in Current Protocols in Immunology (Coligan J. E., et al., eds.), John Wiley & Sons, New York (2003), pages 2.8.1 2.8.10 and 2.10A.1 2.10A.5. Other methods of cleaving antibodies, such as separating heavy chains to form monovalent light and heavy chain fragments (Fd), further cleavage of the fragments, or other enzymatic, chemical, or genetic techniques, can also be used so long as the fragments bind to the antigen recognized by the intact antibody.

[0110] Antibody fragments may also be any synthetic or genetically engineered protein, including, for example, an isolated fragment consisting of a light chain variable region, an "Fv" fragment consisting of heavy and light chain variable regions, and a recombinant single-chain polypeptide molecule in which the light and heavy chain variable regions are linked by a peptide linker (scFv protein).

[0111] Another form of antibody fragment is a peptide containing one or more complementarity-determining regions (CDRs) of an antibody. CDRs (also called "minimal recognition units" or "hypervariable regions") can be incorporated into a molecule by either covalent or non-covalent bonding to make it an antigen-binding protein. CDRs can be obtained by constructing a polynucleotide that encodes the target CDR. Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA from antibody-producing cells as a template (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, 1991; Courtenay Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc. 1995)).

[0112] Thus, in one embodiment, the binding agent comprises at least one CDR described herein. The binding agent may comprise at least two, three, four, five, or six CDRs described herein. The binding agent may further comprise at least one variable region domain of an antibody described herein. The variable region domain may be of any size or amino acid composition and generally comprises at least one CDR sequence involved in binding to human M6PR, e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, specifically described herein, adjacent to or in frame with one or more framework sequences. Generally speaking, a variable (V) region domain is a CDR sequence of an immunoglobulin heavy chain (V H ) and / or light chain (V L ) variable domains can be any suitable arrangement of V region domains. Thus, for example, the V region domains can be monomeric and can independently bind to human M6PR with affinities in the range of 1 nM to 1 pM. H or V L Alternatively, the V region domain may be a dimer, H V H , V H V L , or V L V L A V region dimer may comprise at least one V region that may be non-covalently linked. H chain and at least one V L chain (hereinafter, F V Optionally, the chains can be covalently linked, for example, directly via a disulfide bond between the two variable domains, or via a linker (e.g., a peptide linker), to form a single-chain Fv (scFv).

[0113] The variable region domain can be any naturally occurring variable domain or an engineered version thereof. By engineered version is meant a variable region domain created using recombinant DNA engineering techniques. Such engineered versions include, for example, those created from a particular antibody variable region by insertions, deletions, or changes in or to the amino acid sequence of that particular antibody. A particular example is an engineered variable region domain containing at least one CDR and optionally one or more framework amino acids from a first antibody and the remainder of the variable region domain from a second antibody.

[0114] The variable region domain may be covalently linked at the C-terminal amino acid to at least one other antibody domain or fragment thereof. Thus, for example, a V present in the variable region domain may be covalently linked to at least one other antibody domain or fragment thereof. H The V domain may be linked to an immunoglobulin CH1 domain or a fragment thereof. L The domain can be linked to a CK domain or fragment thereof. Thus, for example, an antibody can be constructed in which the antigen-binding domain is covalently linked at its C-terminus to the relevant V domain and to the CH1 domain and CK domain, respectively. H Domain and V L The CH1 domain may be an Fab fragment containing the CH1 domain, which may be extended with additional amino acids, for example to provide the hinge region or part of the hinge region domain found in an Fab' fragment, or to provide additional domains such as antibody CH2 and CH3 domains.

[0115] As described herein, an antibody comprises at least one of these CDRs. For example, one or more CDRs can be incorporated into a known antibody framework region (e.g., IgG1, IgG2, etc.) or conjugated to a suitable vehicle to extend its half-life. Suitable vehicles include, but are not limited to, Fc, polyethylene glycol (PEG), albumin, transferrin, etc. These and other suitable vehicles are known in the art. Such conjugated CDR peptides may be in the form of a monomer, dimer, tetramer, or other forms. In one embodiment, one or more water-soluble polymers are conjugated at one or more specific positions of the binder, for example, at the amino terminus.

[0116] 6.5. Antigen-binding proteins In one aspect, the present invention provides antigen binding proteins (e.g., antibodies, antibody fragments, antibody derivatives, antibody muteins, and antibody variants) that bind to internalizing receptors, soluble target molecules, and / or cell surface target molecules.

[0117] In some embodiments, the ABP comprises an antigen-binding moiety (e.g., at least one antigen-binding moiety) that specifically binds to an antigen of an internalizing receptor. In certain embodiments, antigen binding promotes intracellular transport of the ABP by the internalizing receptor.

[0118] In certain embodiments, the first antigen-binding moiety specifically binds to an epitope on an antigen that promotes intracellular transport of the ABP by an internalization receptor. In certain embodiments, the internalization receptor is a cell surface internalization receptor. In certain embodiments, the internalization receptor is an endocytosis receptor. In certain embodiments, the internalization receptor can target lysosomes. In certain embodiments, the internalization receptor is an oncogenic receptor.

[0119] In certain embodiments, the internalization receptor is CI-M6PR.

[0120] In some embodiments, the antigen-binding portion is an antigen-binding fragment selected from the group consisting of a Fab, a F(ab')2, a single chain antibody (scFv), an (scFv)2, a diabody, a triabody, a tetrabody, and a domain antibody.

[0121] In some embodiments, the antigen-binding portion comprises a Fab fragment and an Fc domain. In some embodiments, a "Fab-Fc" fragment comprises a Fab fragment attached to an Fc domain. For example, the Fc domain may be attached to the C-terminus of the Fab fragment. Any suitable Fc domain known in the art or described herein can be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[0122] In another aspect of the disclosure, the ABP comprises a multispecific ABP. In some embodiments, the multispecific ABP of the disclosure comprises a first antigen-binding portion and a second antigen-binding portion.

[0123] In some embodiments, the multispecific ABP comprises a first antigen-binding moiety that specifically binds to an antigen of an internalizing receptor, and in certain embodiments, antigen binding promotes intracellular transport of the multispecific ABP by the internalizing receptor.

[0124] In certain embodiments, the first antigen-binding moiety specifically binds to an epitope on the antigen that promotes intracellular transport of the multispecific ABP by an internalization receptor. In certain embodiments, the specific epitope provides a function favorable for internalization. In certain embodiments, the internalization receptor is a cell surface internalization receptor. In certain embodiments, the internalization receptor is an endocytosis receptor. In certain embodiments, the internalization receptor can target lysosomes. In certain embodiments, the internalization receptor is an oncogenic receptor.

[0125] In some embodiments, the multispecific ABP binds to an antigen of an internalizing receptor, and the internalizing receptor is a cation-dependent mannose-6-phosphate receptor (CD-M6PR).

[0126] In certain embodiments, the internalizing receptor is CD-M6PR. In certain embodiments, the internalizing receptor is CI-M6PR.

[0127] In certain embodiments, the internalization receptor is CI-M6PR. In some embodiments, the binding of the antigen-binding moiety that binds to CI-M6PR is an allosteric binder and / or a direct conformational agonist that rapidly promotes internalization of M6PR. In some embodiments, the binding of the antigen-binding moiety to CI-M6PR promotes the binding of M6PR to M6P (e.g., competitive and / or non-competitive binding).

[0128] In some embodiments, the first antigen-binding portion comprises an antigen-binding fragment selected from the group consisting of a Fab, a F(ab')2, a single chain antibody (scFv), an (scFv)2, a diabody, a triabody, a tetrabody, and a domain antibody.

[0129] In some embodiments, the antigen-binding portion comprises a Fab fragment and an Fc domain. In some embodiments, a "Fab-Fc" fragment comprises a Fab fragment attached to an Fc domain. For example, the Fc domain may be attached to the C-terminus of the Fab fragment. Any suitable Fc domain known in the art or described herein can be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[0130] In certain embodiments, the multispecific ABP comprises a second antigen-binding moiety that specifically binds to an antigen of a soluble or cell-surface target molecule. In some embodiments, internalization of the soluble or cell-surface target molecule provides a therapeutic effect.

[0131] In certain embodiments, the second antigen-binding moiety specifically binds to a soluble target moiety. In some embodiments, the soluble target molecule is immunoglobulin E (IgE). Non-limiting examples of antigen-binding moieties that bind to IgE can be found in U.S. Patent Nos. 8,071,097 and 7,867,494, and PCT Patent Publication No. WO2017211928A1, which are incorporated herein by reference in their entireties.

[0132] In some embodiments, the second antigen-binding portion comprises a second variable light chain region (VLC) having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 138. L In some embodiments, the second antigen-binding portion comprises a second variable light chain region (V) CDR1 having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 139. L In certain embodiments, the second antigen-binding portion comprises a second variable light chain region (V) CDR2 having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 140. L ) including CDR3.

[0133] In some embodiments, the second antigen-binding portion comprises a second variable heavy chain region (VH) having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 135. HIn some embodiments, the second antigen-binding portion comprises a second variable heavy chain region (V) CDR1 having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 136. H In some embodiments, the second antigen-binding portion comprises a second variable heavy chain region (V) CDR2 having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 137. H ) including CDR3.

[0134] In some embodiments, the second antigen-binding portion comprises a variable light chain (V) having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 134. L In some embodiments, the second antigen-binding portion comprises a variable heavy chain (V) having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 133. H ) is included.

[0135] In certain embodiments, the second antigen-binding moiety specifically binds to a cell surface target molecule. In certain embodiments, the cell surface target molecule is an ERBB receptor (e.g., EGFR, ERBB2, ERBB3, ERBB4), erythropoietin-producing hepatocyte (EPH) receptor, fibroblast growth factor (FGF) receptor (e.g., FGFR1, FGFR2, FGFR3, FGFR4, FGFR5), platelet-derived growth factor (PDGF) receptor (e.g., PDGFR-A, PDGFR-B), vascular endothelial growth factor (VEGF) receptor (e.g., VEGFR1 / FLT1, VEGFR2 / FLK1, VEGF3), tyrosine kinase receptor with immunoglobulin-like and EGF-like domains (TIE) receptor, insulin-like growth factor (IGF) receptor (e.g., INS-R, IGFIR, IR-R), discoidin domain (DD) receptor, c-Met receptor (MET), récepteur d'origine nantais (RON) (also known as macrophage-stimulating 1 receptor), Flt3 The receptor is selected from fins-related tyrosine kinase 3 (Flt3), colony-stimulating factor 1 (CSF1) receptor, adhesion-associated kinase receptor (e.g., Axl), c-kit receptor (KIT), and insulin receptor-related (IRR) receptor.

[0136] In certain embodiments, the cell surface target molecule is epidermal growth factor receptor (EGFR). Non-limiting examples of antigen-binding moieties that bind to EGFR are described in U.S. Patent Nos. 10,954,286, 10,844,127, and 11,040,111, which are incorporated by reference in their entireties.

[0137] In some embodiments, the second antigen-binding portion comprises a second variable light chain region (VLCD) having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 122 and SEQ ID NO: 130. LIn some embodiments, the second antigen-binding portion comprises a second V CDR1 having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 123 and SEQ ID NO: 131. L In certain embodiments, the second antigen-binding portion comprises a second V CDR2 having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 124 and SEQ ID NO: 132. L In certain embodiments, the second antigen-binding portion comprises a second variable heavy chain region (VH) having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 119 and SEQ ID NO: 127. H ) including CDR1.

[0138] In certain embodiments, the second antigen-binding portion comprises a second V having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO:120 and SEQ ID NO:128. H In certain embodiments, the second antigen-binding portion comprises a second V CDR2 having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 121 and SEQ ID NO: 129. H Includes CDR3.

[0139] In some embodiments, the second antigen-binding portion comprises a variable light chain (V) having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO:118 and SEQ ID NO:126. L In some embodiments, the second antigen-binding portion comprises a second variable heavy chain (V) having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 117 and SEQ ID NO: 125. H ) is included.

[0140] In some embodiments, the second antigen-binding portion comprises an antigen-binding fragment selected from the group consisting of a Fab, a F(ab')2, a single chain antibody (scFv), an (scFv)2, a diabody, a triabody, a tetrabody, and a domain antibody.

[0141] An antigen-binding protein may have the structure of, for example, a naturally occurring immunoglobulin. An "immunoglobulin" is a tetrameric molecule. In naturally occurring immunoglobulins, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light chain" (approximately 25 kDa) and one "heavy chain" (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 10 or more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)), incorporated by reference in its entirety for all purposes. The variable regions of each light / heavy chain pair form the antibody binding site; as a result, an intact immunoglobulin has two binding sites.

[0142] Antigen-binding proteins according to the present invention include antigen-binding proteins that promote intracellular transport of ABPs by internalizing receptors. Antigen-binding proteins according to the present invention also include multispecific ABPs that contain a second antigen-binding protein that internalizes a soluble or cell-surface target molecule to provide a therapeutic effect.

[0143] Different antigen-binding proteins may bind to different domains of internalizing receptors, soluble target molecules, or cell surface target molecules via different mechanisms of action. As specifically indicated herein, a domain region is designated to encompass the group unless otherwise indicated. For example, amino acids 4-12 refers to nine amino acids, i.e., the amino acids at positions 4 and 12, as well as the seven intervening amino acids in the sequence.

[0144] ABP derivatives within the scope of the present invention include covalent or aggregative conjugates of ABP or fragments thereof with other proteins or polypeptides, e.g., by expression of recombinant fusion proteins containing heterologous polypeptides fused to the N- or C-terminus of the ABP polypeptide. For example, the conjugated peptide may be a heterologous signal (or leader) polypeptide (e.g., the yeast alpha-factor leader) or a peptide such as an epitope tag. Fusion proteins containing antigen-binding proteins can include additional peptides to facilitate purification or identification of the antigen-binding protein (e.g., poly-His). Antigen-binding proteins can also be linked to the FLAG peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK), as described by Hopp et al., Bio / Technology 6:1204, 1988 and U.S. Patent No. 5,011,912. The FLAG peptide is highly antigenic and provides an epitope that is reversibly bound by specific monoclonal antibodies (mAbs), allowing for rapid analysis and easy purification of expressed recombinant proteins. Reagents useful for preparing fusion proteins in which the FLAG peptide is fused to a given polypeptide are commercially available (Sigma, St. Louis, MO).

[0145] One suitable Fc polypeptide, described in PCT application WO 93 / 10151 (incorporated herein by reference), is a single-chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgG1 antibody. Another useful Fc polypeptide is the Fc mutein described in U.S. Pat. No. 5,457,035 and Baum et al., 1994, EMBO J. 13:3992-4001. The amino acid sequence of this mutein is identical to the native Fc sequence presented in WO 93 / 10151, except that amino acid 19 is changed from Leu to Ala, amino acid 20 is changed from Leu to Glu, and amino acid 22 is changed from Gly to Ala. The mutein exhibits reduced affinity for Fc receptors.

[0146] In some embodiments, the Fc region comprises human IgG1, human IgG2, human IgG4, or a variant thereof. In some embodiments, the Fc region comprises a modification that affects effector function. In some embodiments, the Fc region comprises a LALAPG, N297A, DANA, LALA, or N297Q mutation. A LALAPG mutation can refer to L234A, L235A, and P329G mutations in the Fc region. A DANA mutation can refer to D265A and N297A mutations in the Fc region. A LALA mutation can refer to L234A and L235A mutations in the Fc region.

[0147] In some embodiments, a first antigen-binding portion of the multispecific ABP is in Fab format and a second antigen-binding portion of the multispecific ABP is in scFv format, or a first antigen-binding portion is in scFv format and a second antigen-binding portion is in Fab format (see, e.g., Figures 1, 2A-2B, and 3A-3B, which show non-limiting examples of anti-M6PR / IgE and anti-M6PR / EGFR multispecific antibodies). In some embodiments, a first antigen-binding portion of the multispecific ABP is in Fab format and a second antigen-binding portion of the multispecific ABP is in Fab format. In some embodiments, a first antigen-binding portion of the multispecific ABP is in scFv format and a second antigen-binding portion of the multispecific ABP is in scFv format.

[0148] In some embodiments, the multispecific ABP comprises a full-length antibody sequence that binds to an internalizing receptor, including the variable light chain amino acid sequence of the internalizing receptor antibody, the variable heavy chain amino acid sequence of the internalizing receptor antibody, and the Fc region of the internalizing receptor antibody, as shown in Figures 2A-2B, and an antigen-binding portion that binds to a soluble or cell-surface target molecule, such as an scFv or Fab region of an antibody that binds to a soluble or cell-surface target molecule.

[0149] In some embodiments, the multispecific ABP comprises a full-length antibody sequence targeting a soluble or cell-surface target molecule, including the variable light amino acid sequence of a soluble or cell-surface target molecule antibody, the variable heavy amino acid sequence of a soluble or cell-surface target molecule antibody, and the Fc region of a soluble or cell-surface target molecule antibody, as shown in Figures 3A-3B, and an antigen-binding portion that binds to an internalizing receptor, for example, an scFv or Fab region of an antibody that binds to an internalizing receptor.

[0150] The antigen-binding proteins (e.g., antibodies, antibody fragments, multispecific antibodies, and antibody derivatives) of the present invention can comprise any constant region known in the art. The light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, such as a human kappa- or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region, such as a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. In one embodiment, the light chain constant region or heavy chain constant region is a fragment, derivative, mutant, or mutein of a naturally occurring constant region.

[0151] Techniques for deriving antibodies of a different subclass or isotype from a desired antibody, i.e., subclass switching, are known. Thus, for example, IgG antibodies can be derived from IgM antibodies, and vice versa. Such techniques allow the preparation of new antibodies that possess the antigen-binding properties of a given antibody (parent antibody) while also exhibiting biological properties associated with a different antibody isotype or subclass than the parent antibody. Recombinant DNA technology can be used. Cloned DNA encoding specific antibody polypeptides, such as DNA encoding the constant domain of an antibody of a desired isotype, can be used in such procedures. See also Lantto et al., 2002, Methods Mol. Biol. 178:303-16.

[0152] In one aspect, the present disclosure provides antigen-binding portions comprising antigen-binding fragments of the internalizing receptors, soluble target molecules, and / or cell surface target molecules of the present invention. Such fragments may consist entirely of antibody-derived sequences or may contain additional sequences. Examples of antigen-binding fragments include Fab, F(ab')2, single-chain antibodies (scFv), (scFv)2, diabodies, triabodies, tetrabodies, and domain antibodies. Other examples are provided in Lunde et al., 2002, Biochem. Soc. Trans. 30:500-06.

[0153] In some embodiments, the present disclosure provides a first antigen-binding portion that is a first antigen-binding fragment and a second antigen-binding portion that is a second antigen-binding fragment.

[0154] Single-chain antibodies (scFv) can be formed by linking a heavy chain variable domain (Fv region) fragment and a light chain variable domain (Fv region) fragment via an amino acid bridge (a short peptide linker, e.g., a synthetic sequence of amino acid residues), resulting in a single polypeptide chain. Such single-chain Fvs (scFvs) consist of two variable domain polypeptides (V L and V H These antibodies have been prepared by fusing DNA encoding a peptide linker between DNA encoding the two variable domains. The resulting polypeptides can fold back on themselves to form antigen-binding monomers or multimers (e.g., dimers, trimers, and tetramers) depending on the length of the flexible linker between the two variable domains (Kortt et al., 1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108; Bird et al., 1988, Science 242:423-26; and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83). Different V L and V HThe polypeptides can be combined to form multimeric scFvs that bind to different epitopes (Kriangkum et al., 2001, Biomol. Eng. 18:31-40). Techniques developed for the production of single-chain antibodies include those described in U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544; and de Graaf et al., 2002, Methods Mol. Biol. 178:379-87. ScFvs comprising the variable domain combinations 45A7, 45A12, 43C8, 12D5, 53B11, 35E2, 06D12, 03C12, 18G4, 02C7, 14A3, 10H5, 06G11, 06H10, 01E5, 21E12, 06F1, 20E12, 21D5, 12A1, 06D1, 07B2, 07E11, 56A2, 01A7, 07A4, 16H4, 08D8, 06C1, 35B2, and 17F11 are encompassed by the present invention.

[0155] Antigen-binding portion that binds to M6PR In one aspect of the disclosure, the ABP comprises an antigen-binding protein comprising at least a first antigen-binding portion that binds to M6PR. In some embodiments, the disclosure provides antigen-binding portions comprising a light chain variable region selected from SEQ ID NOS: 17-32 in Table 6 or a heavy chain variable region selected from SEQ ID NOS: 1-16 in Table 5, and fragments, derivatives, muteins, and variants thereof.

[0156] The positions of the CDRs (underlined) that form part of the antigen-binding site are also shown below, and the framework regions (FRs) are the intervening segments of these variable domain sequences. Both the light chain variable region and the heavy chain variable region have three CDRs (CDR1 to 3) and four FRs (FR1 to 4). The CDR regions of each light chain and heavy chain are also grouped according to the type of antibody. Examples of antigen-binding proteins of the present invention include antigen-binding proteins having a combination of light chain variable domains and heavy chain variable domains selected from the group of antibody clone combinations consisting of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 in Tables 5 to 8.

[0157] In some embodiments, the first antigen-binding portion comprises all six CDR sequences (the three light chain CDRs "LCDR1-LCDR3" and the three heavy chain CDRs "HCDR1-HCDR3") that are identical to one of the clones in the library of internal receptor-binding clones in Tables 5-8. In some embodiments, the first antigen-binding portion comprises three of the six CDR sequences (the three light chain CDRs or the three heavy chain CDRs) that are identical to one of the clones in the library of M6PR-binding clones in Tables 5-8. In some embodiments, the first antigen-binding portion comprises one, two, three, four, or five of the six CDR sequences that are identical to one of the clones in the library of M6PR-binding clones in Tables 5-8.

[0158] In one embodiment, the invention provides a first antigen-binding moiety comprising a light chain variable domain comprising an amino acid sequence that differs by one or more residues from the sequence of a light chain variable domain selected from the group consisting of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 in Table 6, wherein each such sequence difference is independently either a deletion, insertion, or substitution of one amino acid residue. In another embodiment, the light chain variable domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identical to the sequence of a light chain variable domain selected from the group consisting of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 in Table 6. In another embodiment, the light chain variable domain comprises an amino acid sequence encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identical to a nucleotide sequence encoding a light chain variable domain selected from the group consisting of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 in Table 6. In another embodiment, the light chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide encoding a light chain variable domain selected from the group consisting of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 in Table 6.In another embodiment, the light chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide encoding a light chain variable domain selected from the group consisting of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 in Table 6. In another embodiment, the light chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of the light chain polynucleotides of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 in Table 6.

[0159] In another embodiment, the invention provides a first antigen-binding moiety comprising a heavy chain variable domain comprising an amino acid sequence that differs by one or more residues from the sequence of a heavy chain variable domain selected from the group consisting of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 in Table 5, wherein each such sequence difference is independently either a deletion, insertion, or substitution of one amino acid residue. In another embodiment, the heavy chain variable domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of a heavy chain variable domain selected from the group consisting of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 in Table 5. In another embodiment, the heavy chain variable domain comprises an amino acid sequence encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence encoding a heavy chain variable domain selected from the group consisting of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 in Table 5. In another embodiment, the heavy chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide encoding a heavy chain variable domain selected from the group consisting of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 in Table 5.In another embodiment, the heavy chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide encoding a heavy chain variable domain selected from the group consisting of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 in Table 5. In another embodiment, the heavy chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a heavy chain polynucleotide disclosed herein.

[0160] In some embodiments, the heavy chain variable domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 1-16. In some embodiments, the light chain variable domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 17-32.

[0161] Certain embodiments of antigen-binding portions of the present disclosure comprise one or more amino acid sequences identical to the amino acid sequences of one or more of the CDRs and / or FRs referenced herein. In one embodiment, the antigen-binding portion comprises the light chain CDR1 (LCDR1) sequence shown above. In another embodiment, the antigen-binding portion comprises the light chain CDR2 (LCDR2) sequence shown above. In another embodiment, the antigen-binding portion comprises the light chain CDR3 (LCDR3) sequence shown above. In another embodiment, the antigen-binding portion comprises the heavy chain CDR1 (HCDR1) sequence shown above. In another embodiment, the antigen-binding portion comprises the heavy chain CDR2 (HCDR2) sequence shown above. In another embodiment, the antigen-binding portion comprises the heavy chain CDR3 (HCDR3) sequence shown above.

[0162] In one embodiment, the invention provides antigen-binding portions comprising one or more CDR sequences that differ from the above CDR sequences by no more than 5, 4, 3, 2, or 1 amino acid residue.

[0163] In some embodiments, at least one of the HCDR1 sequences of the antigen-binding portion is an HCDR1 sequence from 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 in Table 7. In some embodiments, at least one of the HCDR2 sequences of the antigen-binding portion is an HCDR2 sequence from 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 in Table 7. In some embodiments, at least one of the HCDR3 sequences of the antigen-binding portion is an HCDR3 sequence from 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 in Table 7.

[0164] In some embodiments, the antigen-binding portion comprises a variable heavy chain region (VH) having an amino acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 96%, at least 98%, at least 99%, or at least 100% identical to an amino acid sequence selected from SEQ ID NOs: 33, 36, 39, 42, 45, 47, 50, 56, 61, 64, 67, and 70. H In some embodiments, the antigen-binding portion comprises a V CDR1 having an amino acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 96%, at least 98%, at least 99%, or at least 100% identical to an amino acid sequence selected from SEQ ID NOs: 34, 37, 40, 43, 48, 51, 53, 57, 59, 62, 65, 68, and 71. HIn some embodiments, the antigen-binding portion comprises a V CDR2 having an amino acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 96%, at least 98%, at least 99%, or at least 100% identical to an amino acid sequence selected from SEQ ID NOs: 35, 38, 41, 44, 46, 49, 52, 54, 55, 58, 60, 63, 66, 69, 72, and 73. H Includes CDR3.

[0165] In some embodiments, the antigen-binding portion comprises a variable heavy chain region (VH) having an amino acid sequence at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 33, 36, 39, 42, 45, 47, 50, 56, 61, 64, 67, and 70. H In some embodiments, the antigen-binding portion comprises a V CDR1 having an amino acid sequence at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 34, 37, 40, 43, 48, 51, 53, 57, 59, 62, 65, 68, and 71. H In some embodiments, the antigen-binding portion comprises a V CDR2 having an amino acid sequence at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 35, 38, 41, 44, 46, 49, 52, 54, 55, 58, 60, 63, 66, 69, 72, and 73. H Includes CDR3.

[0166] In another embodiment, the light chain LCDR1 sequence of the antigen-binding portion is a light chain LCDR1 sequence from 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 in Table 8. In some embodiments, the light chain LCDR2 sequence of the antigen-binding portion is a light chain LCDR2 sequence from 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 in Table 8. In some embodiments, the light chain LCDR3 sequence of the antigen-binding portion is a light chain LCDR3 sequence from 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 in Table 8.

[0167] In some embodiments, the antigen-binding portion comprises a variable light chain region (VLC) having an amino acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 96%, at least 98%, at least 99%, or at least 100% identical to an amino acid sequence selected from SEQ ID NOs: 74, 77, 79, 82, 86, 89, 92, 95, 97, 100, 102, 105, 108, 111, and 114. L In some embodiments, the antigen-binding portion comprises a V CDR1 having an amino acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 96%, at least 98%, at least 99%, or at least 100% identical to an amino acid sequence selected from SEQ ID NOs: 75, 80, 83, 87, 90, 93, 98, 103, 106, 109, 112, and 115. LIn some embodiments, the antigen-binding portion comprises a V CDR2 having an amino acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 96%, at least 98%, at least 99%, or at least 100% identical to an amino acid sequence selected from SEQ ID NOs: 76, 78, 81, 84, 85, 88, 91, 94, 96, 99, 101, 104, 107, 110, 113, and 116. L Includes CDR3.

[0168] In some embodiments, the antigen-binding portion comprises a variable light chain region (VLC) having an amino acid sequence at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 74, 77, 79, 82, 86, 89, 92, 95, 97, 100, 102, 105, 108, 111, and 114. L In some embodiments, the antigen-binding portion comprises a V CDR1 having an amino acid sequence at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 75, 80, 83, 87, 90, 93, 98, 103, 106, 109, 112, and 115. L In some embodiments, the antigen-binding portion comprises a V CDR2 having an amino acid sequence at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 76, 78, 81, 84, 85, 88, 91, 94, 96, 99, 101, 104, 107, 110, 113, and 116. L Includes CDR3.

[0169] In another embodiment, the antigen binding portion comprises 1, 2, 3, 4, or 5 CDR sequence(s), wherein each CDR sequence independently differs from the CDR sequences of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 by the addition, substitution, and / or deletion of 6, 5, 4, 3, 2, 1, or 0 single amino acid(s), and the antigen binding protein further comprises 1, 2, 3, 4, or 5 CDR sequence(s), wherein each CDR sequence independently differs from the CDR sequences of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 by the addition, substitution, and / or deletion of 6, 5, 4, 3, 2, 1, or 0 single amino acid(s). In some embodiments, the antigen-binding portion comprises one, two, three, four, or five CDR sequence(s) each having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the CDR sequences of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11.

[0170] The nucleotide sequence of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11, or the amino acid sequence of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11, can be modified by random mutagenesis or site-specific mutagenesis (e.g., oligonucleotide-directed site-directed mutagenesis) to generate modified polynucleotides that contain one or more specific nucleotide substitutions, deletions, or insertions compared to the unmutated polynucleotide. Examples of techniques for making such modifications are described in Walder et al., 1986, Gene 42:133; Bauer et al., 1985, Gene 37:73; Craik, BioTechniques, January 1985, 12-19; Smith et al., 1981, Genetic Engineering: Principles and Methods, Plenum Press, and U.S. Patent Nos. 4,518,584 and 4,737,462. These and other methods can be used to generate derivatives of anti-internalizing receptor antibodies that have desired properties (e.g., improved affinity, avidity, or specificity for the internalizing receptor, improved in vivo or in vitro activity or stability, or reduced in vivo side effects) compared to, for example, the underivatized antibody.

[0171] In one embodiment, an antigen-binding portion of the invention comprises the IgG1 heavy chain domain of any of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11, or a fragment of the IgG1 heavy chain domain of any of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11. In another embodiment, an antigen binding portion of the invention comprises the kappa light chain constant region of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11, or a fragment of the kappa light chain constant region of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11. In another embodiment, an antigen-binding portion of the invention comprises both the IgG1 heavy chain domain of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11, or a fragment thereof, and the kappa light chain domain of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11, or a fragment thereof.

[0172] In some embodiments, at least one antigen-binding portion of the invention includes, for example, variable domain combinations 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 having a desired isotype (e.g., IgA, IgG1, IgG2, IgG3, IgG4, IgM, IgE, and IgD), and those comprising Fab or F(ab')2 fragments thereof. Furthermore, if IgG4 is desired, it may be desirable to introduce a point mutation (CPSCP to CPPCP) in the hinge region, as described in Bloom et al., 1997, Protein Science 6:407 (incorporated herein by reference), to reduce the tendency of IgG4 antibodies to form intra-H chain disulfide bonds, which can lead to heterogeneity.

[0173] In one embodiment, the antigen-binding portion of the antigen-binding protein is 1x10 -4 s -1 The following K off In another embodiment, K off is 5x10 -5 s -1 In another embodiment, K off is substantially the same as an antibody having a combination of light chain variable domain sequences and heavy chain variable domain sequences selected from the group of combinations consisting of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 in Tables 5-6. In another embodiment, the antigen-binding portion has substantially the same K as an antibody comprising one or more CDRs from an antibody having a combination of light chain variable domain sequences and heavy chain variable domain sequences selected from the group of combinations consisting of 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 in Tables 7-8. offIn another embodiment, the antigen-binding portion binds to M6PR with substantially the same K as an antibody comprising one of the amino acid sequences set forth above. off In another embodiment, the antigen-binding portion binds to M6PR at substantially the same K as an antibody comprising one or more CDRs from an antibody comprising one of the amino acid sequences set forth above. off It binds to M6PR.

[0174] The antigen binding protein of the disclosure comprising the first antigen binding portion has a binding affinity for an internalizing receptor of 5×10 -7 M or less, 1×10 -7 M or less, 0.5×10 -7 M or less, 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less, 1×10 -11 M or less, or 1 x 10 -12 In some embodiments, the first antigen-binding moiety has a K in the range of 1 nM to 1 pM. D binds to internalizing receptors.

[0175] In some embodiments, the first antigen-binding moiety has a K of less than 500 nM. D In some embodiments, the first antigen-binding moiety binds to an internalizing receptor with a K of less than 200 nM. D In some embodiments, the first antigen-binding moiety binds to an internalizing receptor with a K of less than 25 nM. D In some embodiments, the first antigen-binding moiety binds to an internalizing receptor with a K of less than 5 nM. D In some embodiments, the first antigen-binding moiety binds to an internalizing receptor with a K of less than 2 nM. D In some embodiments, the first antigen-binding moiety binds to an internalizing receptor at 1×10 -7 M~5×10 -12 K in the M range D In some embodiments, the first antigen-binding moiety binds to an internalizing receptor at 2×10 -7 M~9.5×10 -8 K in the M range Dbinds to internalizing receptors.

[0176] The affinity of an antibody or antigen-binding protein for a binding partner can be determined by those skilled in the art using conventional techniques, such as those described by Scatchard et al. (Ann. NY Acad. Sci. 51:660-672 (1949)) or by surface plasmon resonance (SPR, BIAcore, Biosensor, Piscataway, NJ). In surface plasmon resonance, a target molecule is immobilized on a solid phase and exposed to a ligand in a mobile phase flowing along a flow cell. Binding of the ligand to the immobilized target changes the local refractive index, leading to a change in the SPR angle, which can be monitored in real time by detecting changes in the intensity of reflected light. The rate of change of the SPR signal can be analyzed to obtain the apparent rate constants for the association and dissociation phases of the binding reaction. The ratio of these values provides the apparent equilibrium constant (affinity) (see, e.g., Wolff et al., Cancer Res. 53:2560-65 (1993)).

[0177] An antibody or antigen-binding protein according to the present disclosure can belong to any immunoglobulin class, e.g., IgG, IgE, IgM, IgD, or IgA, and can be obtained or derived from animals, such as poultry (e.g., chicken) and mammals (including, but not limited to, mice, rats, hamsters, rabbits, or other rodents, cows, horses, sheep, goats, camels, humans, or other primates).

[0178] Monoclonal Antibodies In another aspect, the invention provides monoclonal antibodies that bind to an internalizing receptor (e.g., M6PR). The monoclonal antibodies of the invention can be produced using a variety of known techniques. In general, monoclonal antibodies that bind to a specific antigen can be obtained by methods well known to those skilled in the art (see, for example, Kohler et al., Nature 256:495, 1975; Coligan et al. (eds.), Current Protocols in Immunology, 1:2.5.12.6.7 (John Wiley & Sons 1991); U.S. Patent Nos. RE32,011, 4,902,614, 4,543,439, and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.) (1980); Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press (1988); and Picksley et al., "Production of monoclonal antibodies"). (See, "antibody fragments against proteins expressed in E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford University Press 1995)). Antibody fragments can be derived therefrom using any suitable standard technique, such as proteolytic digestion, or optionally, proteolytic digestion (e.g., using papain or pepsin), followed by mild reduction of disulfide bonds and alkylation. Alternatively, such fragments can be produced by recombinant genetic engineering techniques as described herein.

[0179] Monoclonal antibodies can be obtained by injecting an immunogen containing human M6PR or a fragment thereof into an animal, such as a rat, hamster, rabbit, or preferably a mouse (including, for example, a transgenic or knockout mouse known in the art), according to methods known in the art and described herein. The presence of specific antibody production can be monitored by collecting serum samples after the initial injection and / or after booster injections and detecting the presence of antibodies that bind to human M6PR or the peptide using any one of several immunodetection methods known in the art and described herein. From an animal producing the desired antibodies, lymphoid cells (most commonly cells from the spleen or lymph nodes) are removed to obtain B-lymphocytes. The B lymphocytes are then fused with a drug-sensitized myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal and optionally has other desirable properties (e.g., inability to express endogenous Ig gene products (e.g., P3X63-Ag 8.653 (ATCC No. CRL 1580); NSO, SP20)), to generate immortalized eukaryotic cell lines, called hybridomas.

[0180] Lymphoid (e.g., spleen) cells and myeloma cells can be mixed with a membrane fusion promoter, such as polyethylene glycol or a non-ionic detergent, for several minutes and then seeded at low density in a selective medium that supports the growth of hybridoma cells but not unfused myeloma cells. A preferred selective medium is HAT (hypoxanthine, aminopterin, thymidine). After a sufficient time (usually about 1-2 weeks), colonies of cells can be observed. Single colonies can be isolated, and antibodies produced by the cells can be tested for binding activity to human M6PR using any one of a variety of immunoassays known in the art and described herein. Hybridomas can be cloned (e.g., by limiting dilution cloning or soft agar plaque isolation), and positive clones producing antibodies specific to M6PR are selected and cultured. Monoclonal antibodies from hybridoma cultures can be isolated from the supernatant of the hybridoma culture.

[0181] An alternative method for producing mouse monoclonal antibodies is to inject hybridoma cells into the peritoneal cavity of syngeneic mice, e.g., mice that have been treated (e.g., primed with plistatin) to promote the formation of ascites containing the monoclonal antibody. Monoclonal antibodies can be isolated and purified by a variety of established techniques. Such isolation techniques include affinity chromatography using protein A Sepharose, size exclusion chromatography, and ion exchange chromatography (see, e.g., Coligan at pages 2.7.1-2.7.12 and 2.9.1-2.9.3; Baines et al., "Purification of Immunoglobulin G (IgG)," in Methods in Molecular Biology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)). Monoclonal antibodies can be purified by affinity chromatography using an appropriate ligand selected based on specific properties of the antibody (e.g., heavy or light chain isotype, binding specificity, etc.). Examples of suitable ligands immobilized on a solid support include Protein A, Protein G, constant region (light or heavy chain) antibodies, anti-idiotypic antibodies, and M6PR binding proteins, or fragments or variants thereof.

[0182] The antibodies of the present disclosure may also be humanized or fully human monoclonal antibodies.

[0183] 6.7.pH-dependent antigen binding In one aspect, the present invention provides an antigen-binding protein (ABP) comprising a first antigen-binding moiety that specifically binds to the internalization domain of the cation-independent mannose-6-phosphate receptor (CI-M6PR), which promotes intracellular transport of the ABP, wherein binding of the ABP to CI-M6PR is pH-dependent. In such cases, the first antigen-binding moiety preferentially dissociates from the antigen in intracellular compartments having a lower pH.

[0184] In some embodiments, the first antigen-binding moiety binds to CI-M6PR with higher affinity at pH 7.4 than at pH 6.0. In some embodiments, the first antigen-binding moiety binds to CI-M6PR with higher affinity at pH 7.4 than at pH 5.5. In some embodiments, the first antigen-binding moiety binds to CI-M6PR with higher affinity at pH 7.4 than at pH 5.0.

[0185] In some embodiments, the antigen-binding portion is released from CI-M6PR at pH 7.4 or below. In some embodiments, the antigen-binding portion is released from CI-M6PR at pH 6.0 or below. In some embodiments, the antigen-binding portion is released from CI-M6PR at pH 5.5 or below. In some embodiments, the antigen-binding portion is released from CI-M6PR at pH 5.0 or below.

[0186] In some embodiments, the first antigen-binding moiety has a K in the range of 1, 2, 5, 10, 20, 25, 50, 75, or 100 or more at pH 6.0 / pH 7.4. D Ratio or K off It has a ratio.

[0187] In some embodiments, the first antigen-binding moiety has a K in the range of 1, 2, 5, 10, 20, 25, 50, 75, or 100 or more at pH 5.5 / pH 7.4. D Ratio or K off It has a ratio.

[0188] In some embodiments, the first antigen-binding moiety has a K in the range of 1, 2, 5, 10, 20, 25, 50, 75, or 100 or more at pH 5.0 / pH 7.4. D Ratio or K off It has a ratio.

[0189] 6.8. Internal Migration Domain In some embodiments, the antigen-binding domain specifically binds to the internalization domain of the target molecule. The internalization domain of the target molecule promotes, is involved in, or otherwise contributes to the intracellular transport of the ABP. In some embodiments, the internalization domain of the target molecule promotes, is involved in, or otherwise contributes to the transport of the ABP to the endolysosomal compartment. In some embodiments, when the target molecule is present on the surface of a cell, after promoting, being involved in, or otherwise contributing to the internalization of the ABP, the internalization domain of the target molecule promotes, is involved in, or otherwise contributes to the recycling of the target molecule to the cell surface.

[0190] In some embodiments, when the antigen binding domain specifically binds to the internalization domain of a target molecule, binding activates the target molecule, and the activated target molecule promotes, participates in, or otherwise contributes to the intracellular transport of the ABP.

[0191] 6.9.Target binding moiety In one aspect, the present invention provides an antigen binding protein (ABP) comprising a first antigen-binding moiety that specifically binds to the internalization domain of the cation-independent mannose-6-phosphate receptor (CI-M6PR), which promotes intracellular transport of the ABP, and a target-binding moiety that specifically binds to a target molecule.

[0192] In some embodiments, the ABP is an aptamer that specifically binds to a target molecule such as a target protein. In some embodiments, the ABP is a peptide or protein (e.g., a peptide-binding motif, a protein domain, an engineered polypeptide, or a glycoprotein) that specifically binds to a target molecule such as a target protein. In some embodiments, the ABP is an antibody or antibody fragment that specifically binds to a target molecule such as a target protein. In some embodiments, Y is a polynucleotide or oligonucleotide that specifically binds to a target molecule such as a target protein or a target nucleic acid.

[0193] In some embodiments, peptides or proteins that specifically bind to target molecules include, but are not limited to, peptide binding motifs, protein domains, engineered polypeptides, or glycoproteins.

[0194] In some embodiments, the target-binding moiety is an aptamer that specifically binds to a target molecule. Aptamers are nucleic acid molecules that have specific binding affinity for molecules through interactions other than classical Watson-Crick base pairing. Aptamers specifically bind to selected targets and can modulate the activity of the target (e.g., a binding aptamer may block the target molecule's ability to function). Typical aptamers are 10-15 kDa (30-45 nucleotides) in size, bind to their targets with subnanomolar affinity, and discriminate between closely related targets (e.g., aptamers typically do not bind to other proteins from the same gene family). Aptamers bind to target molecules through interactions (e.g., hydrogen bonding, electrostatic complementarity, hydrophobic contacts, steric exclusion) that promote affinity and specificity.

[0195] In some embodiments, the target binding moiety is a ligand that specifically binds to a target molecule. In some embodiments, the ligand is a molecule that can modulate signal transduction. In some embodiments, the ligand is a cytokine (e.g., a pro-inflammatory cytokine or an anti-inflammatory cytokine). Non-limiting examples of cytokines include IL-1 family cytokines (non-limiting examples include IL-1A, IL-1B, IL-18, and IL-33), TNF family cytokines (non-limiting examples include TNF-alpha, TNF-beta, and 4-1BBL), interferon family cytokines (non-limiting examples include IFN-alpha (IFNA), IFN-gamma (IFNG), and IFN-beta (IFNB)), IL-6 family cytokines (non-limiting examples include IL-6, IL-11, and LIF), IL-10 family cytokines (non-limiting examples include IL-10, IL-19, IL-20, IL-22), and TGF-beta family cytokines (e.g., TGF-beta 1, TGF-beta 2, and TGF-beta 3).

[0196] In some embodiments, the target binding moiety is a second antigen binding moiety that specifically binds to a different target molecule than the first antigen binding moiety.

[0197] In some embodiments, the target binding moiety is a second antigen binding moiety that specifically binds to the same target molecule as the first antigen binding moiety. In such cases, the second antigen binding domain can be any of the second antigen binding domains described herein. In some embodiments, the target binding moiety is a second antigen binding moiety that specifically binds to the same target molecule as the first antigen binding moiety, but to a different epitope.

[0198] In some embodiments, the target binding moiety is a small molecule.

[0199] In some embodiments, the target binding moiety is attached to the first antigen binding moiety, optionally via a linker (e.g., any of the linkers described herein). In some embodiments, the target binding moiety is attached to the first antigen binding moiety using a linker (e.g., any of the linkers described herein). In some embodiments, the linker is selected based on the type of target binding moiety.

[0200] In some embodiments, the target binding moiety is attached to the first antigen binding moiety using a linker selected as described in U.S. Pat. Nos. 7,837,980 B2, 8,906,376 B2, 10,201,615 B2, 10,933,112 B2, U.S. Patent Publication Nos. 2019 / 0290775 A1, 2022 / 0111066 A1, and PCT Publication No. WO 2021 / 259506 A1.

[0201] In some embodiments where the target-binding moiety is a small molecule, the small molecule can be attached to the antigen-binding protein at an activatable site. Suitable activatable sites include attachment points such as thiol groups, amino groups (e.g., the epsilon-amino group of a lysine residue or at the N-terminus of a protein), vicinal hydroxyl groups (1,2-diols) (e.g., oxidized carbohydrates), and carboxyl groups (e.g., the C-terminus of a protein, carbohydrates such as aspartic acid and glutamic acid residues, and sialic acid residues).

[0202] In some embodiments where the target-binding moiety is a small molecule, the small molecule can be directly attached to an attachment point on the antigen-binding protein. For example, drugs can be attached by alkylation of the ε-amino group of antibody lysines, reductive amination of oxidized carbohydrates or reaction with hydrazides, transesterification between hydroxyl and carboxyl groups, amidation at amino or carboxyl groups, and attachment to thiols (e.g., interchain thiols) or introduced thiols, for example, by alkylating lysines with 2-iminothiolane. Suitable methods for attaching drugs to attachment points are disclosed, for example, in Current Protocols in Protein Science (John Wiley & Sons, Inc.), Chapter 15: Chemical Modifications of Proteins, which is incorporated herein by reference in its entirety.

[0203] In some embodiments where the target-binding moiety is a small molecule, the small molecule can be indirectly attached to the antigen-binding protein via another molecule, such as a linker. For example, a drug can be attached via a maleimide group attached to a sulfhydryl group, for example, in the hinge region of an antibody. In some embodiments, an antigen-binding protein (e.g., antibody) conjugate can be made by reacting a maleimide-derivatized form of the drug with an antigen-binding domain (e.g., antibody). In some embodiments, an antigen-binding protein (e.g., antibody) conjugate can be made by reducing an antigen-binding domain (e.g., antibody) to produce a reduced antibody, producing an amine drug, derivatizing the amine drug with a maleimide to produce a maleimide-derivatized drug, and reacting the maleimide-derivatized drug with an antigen-binding domain (e.g., antibody).

[0204] In some embodiments where the target binding moiety is a small molecule, two or more small molecules can be attached (eg, directly or indirectly) to the antigen binding protein.

[0205] 6.9.1.Target molecules In some embodiments, where the ABP comprises a target binding moiety that specifically binds to a target molecule, the target molecule is selected from a soluble extracellular target molecule, a cell surface target molecule, a toxin, a pathogen, and a therapeutic agent.

[0206] In some embodiments, the target molecule is an immunoglobulin, hi some embodiments, the target molecule is immunoglobulin E (IgE).

[0207] In some embodiments, the target molecule is a receptor of the epidermal growth factor receptor (EGFR) family.

[0208] 6.10. Cargo Section In one aspect, the present invention provides antigen binding proteins (ABPs) comprising a first antigen-binding moiety that specifically binds to the internalization domain of the cation-independent mannose-6-phosphate receptor (CI-M6PR), which promotes intracellular transport of the ABP, and a cargo moiety. In some embodiments, the cargo moiety is fused to the antigen binding protein. In some embodiments, the cargo moiety is fused to the N-terminus of the antigen binding protein. For example, if the ABP comprises a Fab fragment and a cargo moiety, the cargo moiety is fused to the N-terminus of the ABP. In some embodiments, the cargo moiety is fused to the C-terminus of the antigen binding protein. For example, if the ABP comprises a Fab-Fc fragment, and the Fc domain is linked to the C-terminus of the Fab fragment, the cargo moiety is fused to the C-terminus of the Fc domain (e.g., the domains are ordered N-terminus to C-terminus: Fab-Fc-cargo moiety).

[0209] In another aspect, the present invention provides an antigen binding protein (ABP) comprising a first antigen binding moiety that specifically binds to the internalization domain of the cation-independent mannose-6-phosphate receptor (CI-M6PR) that facilitates intracellular trafficking of the ABP, a cargo moiety fused to the antigen binding protein, and a target binding moiety that specifically binds to a target molecule (e.g., any of the target molecules described herein).

[0210] In some embodiments, the cargo moiety is selected from a small molecule, a drug, a dye, a fluorophore, a monosaccharide, a disaccharide, a trisaccharide, a biomolecule, a nanoparticle, and a viral composition.

[0211] In some embodiments, the cargo moiety is a biomolecule selected from a polypeptide, a polynucleotide, an aptamer, a polysaccharide, a glycoprotein, a lipid, an enzyme, an antibody, and an antibody fragment.

[0212] In some embodiments, the cargo moiety is attached to the first antigen-binding moiety, optionally via a linker (e.g., any of the linkers described herein). In some embodiments, the cargo moiety is attached to the first antigen-binding moiety using a linker (e.g., any of the linkers described herein). In some embodiments, the linker is selected based on the type of target-binding moiety.

[0213] Linker The terms "linker," "linking moiety," and "linking group" are used interchangeably and refer to a linking moiety that covalently bonds two or more moieties or compounds, such as a ligand and another moiety of interest. In some cases, the linker is divalent and connects two moieties. In certain cases, the linker is a trivalent or higher polyvalent branched linking group. In some cases, the linker connecting two or more moieties has a linear or branched backbone that is 500 atoms or less (e.g., 400 atoms or less, 300 atoms or less, 200 atoms or less, 100 atoms or less, 80 atoms or less, 60 atoms or less, 50 atoms or less, 40 atoms or less, 30 atoms or less, or 20 atoms or less) in length, e.g., as measured between the two or more moieties. The linking moiety is a covalent bond connecting two groups, or a straight or branched chain of 1 to 500 atoms in length, e.g., about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500 carbon atoms in length, and the linker can be straight, branched, cyclic, or single-atom. In certain cases, 1, 2, 3, 4, 5, or more, 10, or more carbon atoms in the linker backbone can be optionally substituted with heteroatoms, e.g., sulfur, nitrogen, or oxygen heteroatoms. In certain examples, when the linker contains a PEG group, every third atom in that segment of the linker backbone is substituted with oxygen. The bonds between the backbone atoms can be saturated or unsaturated, and typically, there are no more than 1, 2, or 3 unsaturated bonds in the linker backbone. The linker may include one or more substituents, such as alkyl, aryl, or alkenyl groups. Linkers may include, but are not limited to, one or more of oligo(ethylene glycol), ether, thioether, disulfide, amide, carbonate, carbamate, tertiary amine, and alkyl, which may be linear or branched (e.g., methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), etc.). The linker backbone may include a cyclic group, such as an aryl, heterocycle, cycloalkyl group, or heterocyclic group, where two or more atoms, such as 2, 3, or 4 atoms, of the cyclic group are included in the backbone.

[0214] In some embodiments, a "linker" or linking moiety is derived from a molecule having two reactive ends, one for binding to a moiety of interest, such as a biomolecule (e.g., an antibody), and the other for binding to a moiety that binds to the cell surface receptor M6PR. When the moiety of interest is a polypeptide, the polypeptide-binding reactive end of the linker is a site that can be attached to the polypeptide via a cysteine thiol or lysine amine group on the polypeptide, as the case may be, and thus can be a thiol-reactive group such as maleimide or dibromomaleimide, or a thiol-reactive group as defined herein, or an amine-reactive group such as an active ester (e.g., a perfluorophenyl ester or a tetrafluorophenyl ester), or an amine-reactive group as defined herein.

[0215] In certain embodiments of the formulas described herein, the linker comprises one or more straight-chain or branched-chain carbon moieties and / or polyether (e.g., ethylene glycol) moieties (e.g., repeating units of -CHCHO-), and combinations thereof. In certain embodiments, these linkers optionally have amide, urea, or thiourea, carbamate, ester, amino, ether, thioether, sulfhydryl, or other heterofunctional bonds. In certain embodiments, the linker comprises one or more carbon atoms, nitrogen atoms, sulfur atoms, oxygen atoms, and combinations thereof. In certain embodiments, the linker comprises one or more ether bonds, thioether bonds, amine bonds, amide bonds, carbon-carbon bonds, carbon-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds, and combinations thereof. In certain embodiments, the linker comprises a linear structure. In certain embodiments, the linker comprises a branched structure. In certain embodiments, the linker comprises a cyclic structure.

[0216] In certain embodiments, the linker is -Ci_20-alkylene-, -NHCO-Ci_6-alkylene-, -CONH-Ci_6-alkylene-, -NHCi_6-alkylene-, -NHCONH-Ci_6-alkylene-, -NHCSNH-Ci_6-alkylene-, -Ci_6-alkylene-NHCO-, -Ci_6-alkylene-CONH-, -Ci_6-alkylene-NH-, -Ci_6-alkylene-NHCONH-, -Ci_6-alkylene-NHCSNH-, O(CH2)p-, -(OCH2CH2)p-, -NHCO-, -CONH-, -NHSO2-, -SON2NH-, -CO-, -SO2-, - It comprises one or more linking moieties independently selected from O-, -S-, monocyclic heteroaryl (e.g., 1,2,3-triazole), monocyclic aryl (e.g., phenyl, e.g., 1,4-linked phenyl or 1,3-linked phenyl), monocyclic heterocycle (e.g., a pyrrolidine-2,5-dione, piperazine, or piperidine ring as described herein), amino acid residue (natural or unnatural amino acid residue), -NH-, and -NMe-, where each p is independently 1 to 50.

[0217] 6.11.1. Chemoselective Ligation Groups A chemoselective ligation group is a group having a reactive functional group or functional groups that can bond with a compatible group on a second moiety. For example, the chemoselective ligation group (or its precursor) can be one of a pair of groups associated with conjugation chemistries such as azide-alkyne click chemistry, copper-free click chemistry, Staudinger ligation, tetrazine ligation, hydrazine-iso-Picteth-Spengler (HIPS) ligation, cysteine-reactive ligation chemistry (e.g., thiol-maleimide, thiol-haloacetamide, or alkyne hydrothiolation), amine-activated ester coupling, reductive amination, dialkylsquarate chemistry, etc.

[0218] Chemoselective ligation groups that can be utilized in joining two moieties include, but are not limited to, amino (e.g., the N-terminal amino group or lysine side chain group of a polypeptide), azide, arylazide, alkynyl (e.g., ethynyl or cyclooctyne or derivative), active ester (e.g., N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, or PFP ester or thioester), haloacetamide (e.g., iodoacetamide or bromoacetamide), chloroacetyl, bromoacetyl, hydrazide, maleimide, vinyl sulfone, 2-sulfonylpyridine, cyano-alkyne, thiol (e.g., cysteine residue), disulfide or protected thiol, isocyanate, isothiocyanate, aldehyde, ketone, alkoxyamine, hydrazide, aminooxy, phosphine, HIPS hydrazinyl-indolyl group, or aza-HIPS hydrazinyl-pyrrolo-pyridinyl group, tetrazine, cyclooctene, squarate, and the like.

[0219] Conjugates of the present disclosure can be made using a variety of linkers and / or bifunctional protein coupling agents, such as 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, sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate). The present disclosure further contemplates that the conjugates described herein can be prepared using any suitable method disclosed in the art (see, e.g., Bioconjugate Techniques (Hermanson ed., 2d ed. 2008)).

[0220] In some embodiments, the chemoselective ligation group can spontaneously bond to a compatible chemical group when the two groups are contacted under appropriate conditions (e.g., copper-free click chemistry conditions). In some examples, the chemoselective ligation group can bond to a compatible chemical group when the two groups are contacted in the presence of a catalyst or other reagent (e.g., copper-catalyzed click chemistry conditions). In some embodiments, the chemoselective ligation group is a photoactive ligation group. For example, upon irradiation with ultraviolet light, a diazirine group can form a reactive carbene, which can insert into the C-H, N-H, and O-H bonds of a second moiety.

[0221] Arrays Antibodies 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 comprise heavy and light chain V(J)D polynucleotides (e.g., of antibody clones 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11). Antibodies 3C7, 6D1, 6D12, 6F1, 6H10, 8D8, 10H5-2, 11B12, 12D5, 16H4, 17F11, 18G4, 21D5, 43C8, 45A12, and 53B11 comprise the variable light chain and variable heavy chain sequences listed in Tables 5-6. The CDR sequences in the light and heavy chains are also provided by the specific SEQ ID NOs. [Table 5-1] [Table 5-2] [Table 5-3] [Table 6-1] [Table 6-2] [Table 6-3] [Table 7-1] [Table 7-2] [Table 8-1] [Table 8-2] [Table 9] [Table 10-1] [Table 10-2] [Table 11-1] [Table 11-2] [Table 11-3] [Table 12-1] [Table 12-2] [Table 12-3]

[0222] Pharmaceutical Compositions Pharmaceutical compositions comprising the proteins and polypeptides of the invention are also provided, such compositions comprising a therapeutically or prophylactically effective amount of the polypeptide or protein in admixture with pharmaceutically acceptable and physiologically acceptable formulation agents.

[0223] Pharmaceutical compositions may contain formulation substances to alter, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution rate or release rate, adsorption, or penetration of the composition.

[0224] Suitable formulation substances include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrin), proteins (such as serum albumin, gelatin, or immunoglobulins), colorants, flavoring agents, and diluents, emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium). The additives include, but are not limited to, preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as Pluronic®, PEG, sorbitan esters, polysorbates (such as polysorbate 20, polysorbate 80), Triton, tromethamine, lecithin, cholesterol, tyloxapal), stability enhancers (sucrose or sorbitol), tonicity enhancers (such as alkali metal halides (preferably sodium or potassium chloride, mannitol, sorbitol)), delivery vehicles, diluents, excipients, and / or pharmaceutical adjuvants. Neutral buffered saline or saline mixed with conspecific serum albumin are examples of suitable diluents. Preservatives such as benzyl alcohol may also be added in accordance with appropriate industry standards. The composition may be formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose) as diluents. Suitable ingredients are non-toxic to recipients at the dosages and concentrations used.Further examples of ingredients that can be used in pharmaceutical formulations are provided in Remington's Pharmaceutical Sciences, 16th Ed. (1980) and 20th Ed. (2000), Mack Publishing Company, Easton, PA.

[0225] Optionally, the composition further comprises one or more physiologically active agents, e.g., an anti-angiogenic agent, a chemotherapeutic agent (such as capecitabine, 5-fluorouracil, or doxorubicin), an analgesic agent, etc., non-exclusive examples of which are provided herein. In various specific embodiments, the composition comprises one, two, three, four, five, or six physiologically active agents in addition to the internalizing receptor-binding protein, cell surface targeting molecule, and / or soluble targeting molecule.

[0226] In another embodiment of the present invention, the compositions disclosed herein can be formulated in neutral or salt form.Exemplary pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. Once formulated, solutions are administered in a manner compatible with the dosage formulation and in a therapeutically effective amount.

[0227] Carriers can further include any solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to humans.

[0228] The optimal pharmaceutical composition can be determined by those skilled in the art depending on, for example, the intended route of administration, delivery format, and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the polypeptide. For example, suitable compositions can be water for injection or saline for parenteral administration.

[0229] 6.14.How to use In some embodiments, the antigen binding proteins of the present disclosure can be used to treat a subject having a disease or disorder associated with the presence of an excess of cell surface and / or soluble target molecules.

[0230] In some embodiments, the antigen binding proteins of the present disclosure can be used to treat subjects with allergic conditions, including but not limited to allergic asthma, nasal polyps, and hives.

[0231] Therapeutic antibodies that specifically bind to internalizing receptors can be used, as well as therapeutic antibodies that specifically bind to either internalizing receptors and soluble or cell surface target molecules.

[0232] In vivo and / or in vitro assays can be used, as necessary, to identify optimal dosage ranges. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0233] Aspects of the present disclosure include methods of making an isolated multispecific antigen binding protein (ABP) that comprises an antigen-binding portion that specifically binds to an internalizing receptor and promotes intracellular transport of the ABP by the internalizing receptor. In some embodiments, the antigen-binding portion specifically binds to an epitope on an antigen that promotes intracellular transport of the ABP by the internalizing receptor.

[0234] Aspects of the present disclosure provide methods of treating a disease, the methods comprising administering to a subject in need of treatment an effective amount of a multispecific ABP of the present disclosure or a pharmaceutical composition of the present disclosure.

[0235] The compositions can be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition to be treated. For example, the pharmaceutical compositions can be administered in combination with one or more agents that target different soluble or cell surface target molecules. [Example]

[0236] 6.15. Working Example Below are examples of specific embodiments for carrying out the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0237] The practice of the present invention employs, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, within the skill of the art. Such techniques are fully explained in the literature. See, for example, T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); and Carey and Sundberg Advanced Organic Chemistry, 3rd Ed. (Plenum Press), Vols. A and B (1992). Additionally, the antibody generation and selection methods described in Adler et al., A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library, MAbs (2018) and Adler et al., Rare, high-affinity mouse anti-PD-1 antibodies that function in checkpoint blockade, discovered using microfluidics and molecular genomics, MAbs (2017), which are incorporated by reference in their entireties, can be used.

[0238] 6.15.1. Example 1: Generation of anti-M6PR antibodies To generate monoclonal antibodies that specifically bind to the MP6R receptor, we utilized a hybridoma-based antibody discovery approach using recombinant human M6PR antigen and various cell lines to support mouse immunization and antibody screening. Mice were immunized using proprietary hyperimmune DiversimAb / DiverGimAb mice (Abveris). Hybridomas were characterized by flow cytometry, ELISA, and Octet Biolayer Interferometry (BLI), as part of cryopreservation of hybridomas and supernatant samples, and antibody sequencing.

[0239] Materials and Methods Each of the following protocols was used for each of the assays described herein.

[0240] Titer Test (ELISA) Method: Indirect ELISA Protocol (Summary) 1. Coat a high-binding ELISA plate with antigen overnight at 4°C. 2 Aspirate and block wells with 2% BSA in 1X PBS for 1 hour. 3 Aspirate and add antiserum dilution in blocking solution and incubate for 1 hour at room temperature. 4 Wash wells 4 times in 1XPBST. 5 Add HRP-conjugated secondary antibody in blocking solution and incubate at room temperature for 45 minutes. 6 Wash wells 5 times in 1XPBST. 7. Add TMB substrate followed by stop solution. Read at 450 nm. Note: (-)ctrl NMS = normal mouse serum

[0241] Titer Test (Flow) Cohort 1, 2 Method: FACS Staining Protocol (Outline of Key Steps) Seed 100,000 cells per well. 2. Resuspend in 50uL of titrated serum (starting at 1:100 and diluting 3 times for 11 points). 3 Incubate on ice for 1 hour. 4. Resuspend in 50uL of secondary antibody (1:500 anti-muFc-A488 and 1:500 Live / Dead near-infrared viability). 5 Incubate on ice for 30 minutes. 6 Read at least 5000 events on the iQue.

[0242] Primary Screening (Flow) Method: iQue Flow Cytometry (Key Steps) Seed 100k cells per well. 2. Resuspend in 100uL of culture medium. 3 Incubate on ice for 1 hour. 4 Resuspend in 50uL of secondary antibody. 5 Incubate on ice for 30 minutes. 6 Fix in 1% PFA. 7 Read at least 5000 events on the iQue.

[0243] Secondary Screening (Flow) Method: iQue Flow Cytometry (Key Steps) Seed 100k cells per well. 2. Resuspend in 100uL of culture medium. 3 Incubate on ice for 1 hour. 4 Resuspend in 50uL of secondary antibody. 5 Incubate on ice for 30 minutes. 6 Fix in 1% PFA. 7 Read at least 5000 events on the iQue.

[0244] Secondary Screening (ELISA) Method: Indirect ELISA Protocol 1. Coat a high-binding ELISA plate with antigen overnight at 4°C. 2 Aspirate and block wells with 2% BSA in 1X PBS for 1 hour. 3 Aspirate and add supernatant 1:1 to blocking solution and incubate for 1 hour at room temperature. 4 Wash wells 4 times in 1XPBST. 5 Add HRP-conjugated secondary antibody in blocking solution and incubate at room temperature for 45 minutes. 6 Wash wells 5 times in 1XPBST. 7. Add TMB substrate followed by stop solution. Read at 450 nm. (-)ctrl H10 = medium negative control (+)ctrl H11 = Cohort 1 antiserum positive control (+)ctrl H12 = Cohort 2 antiserum positive control

[0245] Characterization of K562 cells for antibody screening Wild-type K562 cells expressing M6PR and M6PR knockout (KO) K562 cells lacking the M6PR gene were used for antibody screening. Cell surface and intracellular staining of human M6PR in WT and M6PR-KO K562 cells is shown in Figure 4.

[0246] Antibodies and fluorescent stains used in the antibody screening assay include anti-M6PR (clone MEM238)-Alexafluor647 (Abcam).

[0247] Cells were frozen and then stained and fixed for cell surface staining. For intracellular staining, cells were fixed, permeabilized, and stained. [Table 13]

[0248] Mouse immunization and titer testing Cohorts of mice were immunized using the reagents listed in Table 15 according to the following method and schedule. The health of the mice was monitored daily. Two cohorts of mice (n=4 / cohort) for DiverSimab and DiverGimAb were each given a priming dose of 100 μg / animal of recombinant human M6PR protein, followed by weekly boosts of 20 μg / animal of recombinant human M6PR protein for 4 weeks. After completion of the immunization schedule, the titers of each mouse were assessed using the recombinant antigen, confirmed by a flow-based assay using human cell lines (K562 WT, K562 M6PR-KO, Hela WT, Hela M6PR-KO), and cross-screened against two mouse cell lines (Dipak MC38 and 4T1) (Figure 5). Mice with high titers were advanced to fusions.

[0249] Hybridoma fusion and antibody screening Tissues, including lymph nodes and / or spleen, were harvested. Hybridomas were seeded and expanded in culture using a high-efficiency electrofusion method, and then high-throughput primary screening of clones (flow cytometry) was performed. To confirm binding, primary screening (flow cytometry) was performed using K562 cells (WT and M6PR-KO). Clones were evaluated by ELISA to assess each clone's binding to the human and mouse M6PR antigen. Clones that tested positive for binding to the human M6PR antigen were advanced to secondary flow-based screening using human cell lines (K562 WT, K562 M6PR-KO, Hela WT, Hela M6PR-KO) and mouse cell lines (Dipak MC38 and 4T1) to confirm specific antibody binding to human M6PR. No mouse cross-reactive monoclonal antibodies were identified; all monoclonal antibodies specifically bound to human M6PR.

[0250] Association / dissociation rates and K for human antigens DThe top 192 clones were advanced to affinity measurement by Octet to measure the V. All 192 clones were sequenced using the Sanger sequencing process to identify the paired V. H / V L Variable region sequences, antibody isotype data, and germline family were generated. Individual anti-mouse biosensors were loaded with mIgG from the supernatant (and a poly-mIgG control) (Figure 6). A baseline was measured for each biosensor. The loaded biosensors were then bound to recombinant His-tagged human M6PR, and the association rate was measured. The biosensors were transferred to buffer wells, and the dissociation rate was measured. Results for a representative clone are shown in Figure 7. Biosensors were regenerated to measure additional analyte interactions (mouse M6PR-His6 and off-target binding to His). Clones that had affinity for human M6PR and showed no binding to the His control and / or mouse M6PR were advanced to binning for determination of epitope diversity.

[0251] 6.15.2. Example 2: Evaluation of anti-CI-M6PR monoclonal antibodies produced by assaying hybridoma supernatants The CI-M6PR-binding antibodies were screened for internalization activity as shown in Figure 8. Because CI-M6PR binds and internalizes IGF2, a non-binding antibody (KLH) fused to IGF2 was used as a positive control for internalization activity. K562 cells were seeded at 30,000 cells per well of a 96-well tissue culture plate in 100 μl RPMI (Gibco #61870143) containing 10% v / v FBS (VWR #89510-188), 2 mM L-alanyl-L-glutamine dipeptide, 100 units / ml penicillin, and 100 μg / ml streptomycin (Gibco #15140148). Separately, 50 μl of CI-M6PR clone hybridoma supernatant was mixed with 50 μl of Zenon pHrodo-iFL IgG labeling reagent (Thermo Fisher #Z25609) and incubated at room temperature for 15 minutes. The hybridoma supernatant / pHrodo mixture was layered on top of K562 cells and incubated for 18 hours to allow for uptake and lysosomal accumulation. Uptake was measured using a Novocyte Advanteon flow cytometer (Agilent Technologies) equipped with a 488 nM laser and FITC detection configuration.

[0252] Table 14 below shows the binding affinities and cell internalization determined for various anti-CI-M6PR monoclonal antibodies according to the methods described above. [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4] [Table 14-5]

[0253] 6.15.3. Example 3: Anti-MP6PR Antibodies Bind to Human M6PR Of the 96 anti-M6PR antibodies identified in Example 2, 31 were isolated, cloned, and recombinantly expressed as monoclonal antibodies.

[0254] Binding affinity and kinetics Each of the 31 antibodies was evaluated for its ability to bind to CI-M6PR. Each of the anti-M6PR antibodies that bound to CI-M6PR was also evaluated for binding affinity and kinetics.

[0255] Anti-M6PR antibody binding to human CI-M6PR was measured by biolayer interferometry (BLI) using a GatorPrime instrument. Anti-M6PR antibodies were prepared in PBS buffer supplemented with 0.002% Tween®-20, 0.02% BSA, and 0.005% NaN3 and immobilized on a sensor coated with anti-mouse IgG Fc antibody. The immobilized anti-M6PR antibodies were then incubated with various concentrations of purified recombinant CI-M6PR (domains 1–9) protein prepared in the same buffer. The reaction plate was maintained at 30°C and shaken at 1,000 rpm throughout the experiment. Data were fitted using GatorOne Software v2.10, and the reaction curves were aligned in a correlation step. The processed association and dissociation curves were globally fitted using a 1:1 binding model to obtain the rate constants for each antibody.

[0256] The ELISA results in Figures 9A to 9G show that 16 of the 31 anti-M6PR antibodies bound to human M6PR.

[0257] The results shown in Figure 10 show that the top 15 anti-M6PR antibodies have various affinities (K D ) and kinetics (K on , K. off ) indicates that

[0258] Epitope binning and domain mapping Each of the 16 anti-M6PR antibodies identified as binding to human CI-M6PR (Figures 9A-9G) was evaluated by epitope binning and domain mapping to determine where each antibody binds to CI-M6PR (Figure 11).

[0259] To determine epitope bins, anti-M6PR antibodies and CI-M6PR proteins were immobilized on an anti-mouse IgG Fc sensor according to the method described above. These probes were then incubated with a second anti-M6PR antibody. Any antibody that resulted in a further increase in the signal detected by the sensor was determined to be in a different epitope bin from the antibody already immobilized on the sensor. Any antibody that resulted in a decrease in signal was determined to be in the same epitope bin as the antibody already immobilized on the sensor. See Figure 12A for a non-limiting description of the assay, and Figure 12B for an exemplary plot showing the signal shifts of different epitope bins. As summarized in Figure 13, 12 of the preferred anti-M6PR antibodies represent eight distinct epitope bins.

[0260] To perform domain mapping of the anti-M6PR antibody, we cloned and purified a series of chimeric human / mouse CI-M6PR constructs. Each of the nine domains (D1–D9) of human CI-M6PR was individually replaced with the equivalent domain from mouse CI-M6PR for all nine domains. Each chimeric construct was tagged at the N-terminus with a HIS tag.

[0261] Next, each of the nine HIS-tagged CI-M6PR domains was recombinantly produced via HEK293 expression of the ectodomain. Figure 14 shows the expression of each of the HIS-tagged D1-D9 CI-M6PR domains. The recombinantly expressed HIS-tagged D1-D9 CI-M6PR domains were purified by collecting HEK293 medium and using a HisTrap affinity column. All domains were purified as non-aggregated monovalent entities of approximately 200 kDa. Recovery was transient, at approximately 1-2 mg / mL.

[0262] BLI experiments were performed to test the binding of each anti-M6PR antibody to each chimeric CI-M6PR protein, according to the method described above. Wild-type human CI-M6PR was used as a positive control. If replacing a specific domain of human CI-M6PR with a domain from mouse CI-M6PR resulted in a loss of binding, that domain was identified as the domain of human CI-M6PR to which a given antibody binds.

[0263] As shown in Figures 15A-15B and 15D-15H, the anti-M6PR antibody domains were mapped for clones 3C7, 6D1, 11B12, 17F11, 18G4, 21D5, and 53B11. Clone 8D8 (Figure 15C) showed similar binding to each of the chimeric CI-M6PR proteins, meaning that the domain of human CI-M6PR to which the clone 8D8 antibody binds could not be determined using this assay.

[0264] A schematic diagram summarizing the domain mapping results of the anti-M6PR antibody clones 3C7, 6D1, 11B12, 17F11, 18G4, 21D5, and 53B11 is shown in Figure 16. For example, 53B11 binds to domain 1 on CI-M6PR, 11B12 binds to domain 4 on CI-M6PR, 18G4 binds to domain 5 on CI-M6PR, 3C7 and 6D1 bind to domain 6 on CI-M6PR, 17F11 binds to domain 7 on CI-M6PR, and 21D5 binds to domain 8 on CI-M6PR.

[0265] 6.15.4. Example 4: Evaluation of pH-dependent dissociation of anti-M6PR Ab from CI-M6PR Anti-M6PR antibodies were immobilized on sensors coated with anti-mouse IgG1 Fc according to the method described above and incubated with human CI-M6PR protein prepared in citrate buffer, pH 7.4. For dissociation, the sensors were transferred to wells containing citrate buffer at pH 7.4, pH 6.0, or pH 5.0 (see Figure 17A). Figures 17B-17D show the binding curves for clones 17F11 (Figure 17B), 18G4 (Figure 17C), and 21D5 (Figure 17D). Clones 17F11 and 21D5 show stronger pH-dependent dissociation than clone 18G4.

[0266] To quantify pH-dependent dissociation, the K values for each antibody were calculated at pH 5.0 and pH 6.0. off K at pH 7.4 off The fold increase in dissociation rate for each antibody at the indicated pH was determined by dividing by 1. Figure 18 shows that the dissociation rates of antibody clones 17F11 and 21D5 increase at low pH (pH 5.0).

[0267] 6.15.5. Example 5: Cellular Uptake of Anti-M6PR Antibodies pH rodo uptake assay of 31 anti-M6PR antibodies Recombinantly expressed anti-M6PR antibodies were evaluated for uptake activity using the pHrodo uptake assay (Figure 19A). Specifically, mAb and Zenon pHrodo green Fab were precomplexed at 50 nM mAb and 100 nM Zenono pHrodo green iFL (2X final concentration) in 100 μl of RPMI + 10% FBS and 1X penicillin / strep for 30 minutes at room temperature. 100 μl of each mAb:Zenon pHrodo Fab was added to 100 μl of RPMI + 10% FBS and 1X pen / strep containing 100,000 K562 cells. Plates were incubated at 37°C and 5% CO for 24 hours. Cells were pelleted and washed once with FACS wash buffer (PBS + 1% bovine serum albumin (BSA)). Uptake of anti-M6PR antibody was assayed by flow cytometry measuring the pHrodo green signal in the FITC channel.

[0268] Because CI-M6PR binds and internalizes IGF2, a non-binding antibody (KLH) fused to IGF2 was used as a positive control for uptake activity. Antibodies that showed higher uptake activity than the KLH-IGF2 positive control were identified. As shown in Figure 19B, antibody clones 6D12, 6H10, 6D1, 17F11, and 21D5 showed greater uptake than KLH-IGF2.

[0269] Assessment of uptake of anti-M6PR / anti-IgE bispecific antibodies A subset of anti-M6PR antibodies was evaluated for uptake activity using the IgE-pH rodo uptake assay. Specifically, the M6PR-Oma bispecific antibody (BsAb) (Figure 2A) and IgE fluorescently labeled with Alexa Fluor 488 or Alexa Fluor 647 (IgE-AF488 or IgE-AF647) were precomplexed at 200 nM (2X final concentration) in RPMI + 10% FBS and 1X penicillin / strep for 30 minutes at room temperature. A 6X log3 serial dilution was performed, and 100 μl of each BsAb:IgE dilution was added to 100 μl of RPMI + 10% FBS and 1X pen / strep containing 100,000 K562 cells. The plate was incubated at 37°C and 5% CO2 for 24 hours. Cells were pelleted and washed once with FACS wash buffer (PBS + 1% BSA). Uptake of anti-M6PR antibodies was assayed by flow cytometry measuring the signal from AF488 and / or AF647.

[0270] A non-binding antibody (KLH) fused to omalizumab (Oma) was used as a negative control for uptake activity. Oma-16590 (Dar4) bispecific antibody was used as a positive control for uptake activity. Figures 20A-20F show the uptake of anti-M6PR-Oma bispecific antibodies. The results shown in Figures 20A and 20B demonstrate that 6H10-Oma, 6D1-Oma, 8D8-Oma, and 17F11-Oma transport fluorescently labeled IgE (IgE-AF488 or IgE-AF647) into cells. The results shown in Figures 20C-20F demonstrate that 6H10-Oma and 6D1-Oma internalize pHrodo-labeled IgE (IgE-phrodo) into acidic compartments, whereas other M6PR-Oma bispecific antibodies do not. In particular, bispecific antibodies including 6D1-oma, 8D8-oma, 17F11-oma, and 6H10-oma showed IgE uptake.

[0271] Evaluation of anti-M6PR / anti-C5 bispecific antibodies for C5 internalization and uptake To assess whether anti-M6PR antibodies can target and internalize C5, we generated a bispecific antibody with one anti-M6PR binding arm and one anti-C5 binding arm (eculizumab "ECU") and assayed uptake activity using pHrodo Green-labeled C5.

[0272] HeLa cells were seeded at 15,000 cells per well in a 96-well plate and incubated overnight. Anti-M6PR / anti-C5 bispecific antibodies were added at 100 nM in 100 μL of complete growth medium (DMEM + 10% FBS). In a separate 96-well plate, pHrodo Green-labeled C5 was added to the growth medium at 500 nM in 100 μL of complete growth medium, followed by two-fold serial dilutions. The serially diluted C5 was then mixed with anti-M6PR / anti-C5 bispecific antibodies (53B11-ECU, 43C8-ECU, 12D5-ECU, 18G4-ECU) in a final volume of 200 μL, added to HeLa cells, and incubated at 37°C for 18 hours. C5 internalization into lysosomes was measured by flow cytometry. Data were collected in the APC channel, and mean fluorescence intensity was calculated.

[0273] The results shown in Figure 26A (pH rodo) and Figure 26B (AF647) demonstrate that the anti-M6PR / anti-C5 bispecific antibodies (53B11-ECU, 43C8-ECU, 12D5-ECU, and 18G4-ECU) transport fluorescently labeled C5 (C5-AF488 or C5-AF647) into cells.

[0274] 6.15.6. Example 6: Evaluation of Internalization and Degradation of Anti-M6PR Antibodies The Nano-Glo HiBiT Extracellular Detection System (Promega, Cat. No. N2422) and the Nano-Glo HiBiT Lytic Detection System (Promega, Cat. No. N3050) were used to measure cell surface and total depletion of HiBiT-tagged proteins of interest, respectively. Figure 21 shows a schematic diagram of the surface and total HiBit assays, where Span indicates the extent of target (e.g., EGFR) depletion.

[0275] HiBiT Assay Protocol Briefly, HCT116 cells expressing HiBiT-tagged EGFR were seeded at 5,000 cells per well in 100 μL in white tissue culture-treated 96-well plates and grown for 24 hours at 37°C. Matuzumab-anti-M6PR bispecific antibody was diluted in medium to prepare a 3-fold dilution series from 40 to 0.002 nM (2X final concentration) and added to the cells at 100 μL per well, along with a medium-only control.

[0276] The EGFR ligand, EGF, was used as a positive control and assayed in a 3-fold dilution series from 100 to 0.005 nM (2X final concentration). After antibody or control treatment, cells were grown at 37°C for 48 h, and the medium was replaced with 50 μL of fresh medium and 50 μL of either extracellular or dissolved HiBiT detection reagent prepared according to the manufacturer's instructions. Briefly, LgBiT protein and Nano-Glo HiBiT substrate were diluted at 1:100 and 1:50 ratios, respectively, in the corresponding Nano-Glo HiBiT buffer. After 3 min of shaking and 10 min of incubation at ambient temperature, HiBiT signal was detected by measuring luminescence on an Envision plate reader (Perkin Elmer). Dose responses were performed in duplicate and normalized to the medium-only control. IC values were calculated by fitting a four-parameter curve in GraphPad Prism. 50 was determined, and the span was calculated from the difference between the first and last points on the curve.

[0277] result Figure 22A shows the surface depletion (i.e., span) of EGFR for 16 matuzumab-anti-M6PR bispecific antibodies. Figure 22B shows the total depletion (i.e., span) of EGFR for 16 matuzumab-anti-M6PR bispecific antibodies. Figure 22C shows the percentage of surface EGFR activity for exemplary matuzumab-anti-M6PR bispecific antibodies (3C7-matuz, 53B11-matuz). Figure 22D shows the percentage of total EGFR activity for exemplary matuzumab-anti-M6PR bispecific antibodies (3C7-matuz, 53B11-matuz). Taken together, this data demonstrated that the anti-M6PR antibody clones 3C7, 6D1, and 17F11 have subnanomolar potency similar to EGF (positive control) in both EGFR internalization and degradation.

[0278] 6.15.7. Example 7: Evaluation of Anti-M6PR Antibody Recycling After internalization, the recycling of anti-M6PR-omalizumab (M6PR-Oma) bispecific antibody was evaluated using an hIgE uptake assay (see, e.g., Figure 23). Specifically, the M6PR-Oma bispecific antibody was diluted to a 2X final concentration in RPMI + 10% FBS. 100 μl of the M6PR-Oma bispecific antibody was added to 100 μl of RPMI + 10% FBS and 1X pen / strep containing 100,000 K562 cells or 100,000 HepG2 cells. The cells were contacted with the M6PR-Oma bispecific antibody, thereby labeling the cells with the bispecific antibody. After labeling, the cells were washed to remove unbound (unlabeled) M6PR-Oma bispecific antibody. Next, the cells were divided into four conditions, as shown in Figure 23: 0', 1-30', R-30', and R-60'.

[0279] In the 0' condition, cells were incubated with hIgE-Alexa Fluor (AF) 647 ("hIgE-AF647") without first internalizing the M6PR-Oma bispecific antibody. The 0' control cells were then washed, and AF647 was measured using flow cytometry to assess the presence of the M6PR-Oma bispecific antibody on the cell surface.

[0280] In the I-30', R-30', and R-60' conditions, cells were incubated at 37°C for 30 minutes to allow for internalization of the M6PR-Oma bispecific antibody. After the 30-minute incubation, cells were exposed to hIgE-AF647. Cells in the I-30' condition were washed to remove unlabeled hIgE-AF647, and AF647 was measured using flow cytometry to assess the presence of the M6PR-Oma bispecific antibody on the cell surface. To induce recycling, cells in R-30' and R-60' were maintained at 37°C for the remainder of the designated incubation period. Next, cells in R-30' and R-60' were washed to remove unlabeled hIgE-AF647, and AF647 was measured using flow cytometry to assess the presence of the M6PR-Oma bispecific antibody on the cell surface.

[0281] Figure 24A shows the internalization and recycling of M6PR-Oma bispecific antibodies by HepG2 cells, as measured by mean fluorescence intensity (MFI). Recycling of each antibody was assessed following each of the indicated incubation protocols, as described above and shown in Figure 23 . The ability of the bispecific antibodies to internalize and recycle to the surface was assessed by comparing the MFI in the I-30', R-30', and R-60' conditions with the 0' condition (Figure 24B). Based on this analysis, clones 12D5, 21D5, 18G4, and 53B11 exhibited M6PR-Oma bispecific antibody recycling most similar to the 0' baseline condition, indicating that these M6PR-Oma bispecific antibodies possess properties that enable their uptake and recycling in HepG2 cells.

[0282] Figure 25A shows the internalization and recycling of MPR6-Oma bispecific antibodies by K562 cells, as measured by mean fluorescence intensity (MFI). Recycling of each antibody was assessed following each of the indicated incubation protocols, as described above and shown in Figure 23 . The ability of the bispecific antibodies to internalize and recycle to the surface was assessed by comparing the MFI in the I-30', R-30', and R-60' conditions with the 0' condition (Figure 25B). Based on this analysis, clones 12D5, 18G4, 21D5, 43C8, and 53B11 demonstrated M6PR-Oma bispecific antibody recycling most similar to the 0' baseline condition, indicating that these matuzumab-anti-M6PR bispecific antibodies promote uptake and recycling in K562 cells.

[0283] 6.15.8. Example 8: Assessment of HER3 internalization and degradation The Nano-Glo HiBiT extracellular detection system (Promega, catalog number N2422) and the Nano-Glo HiBiT lytic detection system (Promega, catalog number N3050) were used to measure cell surface and total depletion of HiBiT-tagged HER3 (human epidermal growth receptor 3), respectively.

[0284] HiBiT Assay Protocol Briefly, HCC1419 cells (trastuzumab-resistant) expressing HiBiT-tagged HER3 were seeded in 100 μL into tissue culture-treated 96-well plates and grown at 37 °C for 24 h. A 3-fold dilution series of LUM-anti-M6PR bispecific antibody was prepared in medium from 20 to 0.001 nM (2X final concentration) and added to the cells in 100 μL per well, along with a medium-only control. Lumuletuzumab (LUM), a humanized anti-human epidermal growth factor receptor 3 (HER3) monoclonal antibody, and NRG1 (neuregulin 1) antibody were used as positive controls. A non-binding antibody (KLH) fused to LUM was used as a negative control for uptake activity.

[0285] After LUM-anti-M6PR bispecific antibody or control treatment, cells were grown at 37°C for 48 h, and the medium was replaced with 50 μL of fresh medium and 50 μL of either extracellular or dissolved HiBiT detection reagent prepared according to the manufacturer's instructions. Briefly, LgBiT protein and Nano-Glo HiBiT substrate were diluted 1:100 and 1:50 in the corresponding Nano-Glo HiBiT buffer, respectively. After 3 min of shaking and 10 min of incubation at ambient temperature, HiBiT signals were detected by measuring luminescence on an Envision plate reader (Perkin Elmer). Dose responses were performed in duplicate and normalized to the medium-only control. IC values were calculated by fitting a four-parameter curve in GraphPad Prism. 50 was determined, and the span was calculated from the difference between the first and last points on the curve.

[0286] LUM-anti-M6PR-mediated HER3 internalization or degradation was assessed at 48 hours. Cell viability after LUM-anti-M6PR treatment was assessed at 72 hours. The results are shown in Figures 27A-B.

[0287] result Figure 27A shows surface (i.e., span) and total (i.e., span) depletion of HER3 for a subset of LUM-anti-M6PR bispecific antibodies. Figure 27B shows cell viability after treatment with a subset of LUM-anti-M6PR bispecific antibodies. The left panel of Figure 27A shows the percentage of surface HER3 activity for exemplary LUM-anti-M6PR bispecific antibodies (6D1, 3C7, 18G4). The right panel of Figure 27A shows the percentage of total HER3 activity for exemplary LUM-anti-M6PR bispecific antibodies (6D1, 3C7, 18G4). Figure 27B shows cell viability after treatment with exemplary LUM-anti-M6PR bispecific antibodies (6D1, 3C7, 18G4). Collectively, this data demonstrated that scfv LUM-anti-M6PR bispecific antibodies increased HER3 internalization compared to the parent antibody (LUM). Furthermore, the data demonstrate that the scfv LUM-antiM6PR bispecific antibody is effective in reducing cell viability of trastuzumab-resistant cells.

[0288] 7. Incorporation by Reference All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., a Genbank sequence or GeneID entry), patent application, or patent were specifically and individually indicated to be incorporated by reference in its entirety for all purposes. This incorporation-by-reference statement is intended by applicant, pursuant to 37 CFR §1.57(b)(1), to relate to any individual publication, database entry (e.g., a Genbank sequence or GeneID entry), patent application, or patent (each of which is clearly identified pursuant to 37 CFR §1.57(b)(2)), even if such citation is not immediately adjacent to the dedicated incorporation-by-reference statement. The inclusion within the specification of a dedicated incorporation-by-reference statement, if any, does not in any way weaken this general incorporation-by-reference statement. The citation of a reference herein is not intended as an admission that the reference is relevant prior art, nor does it constitute any admission as to the content or date of these publications or documents.

[0289] 8. Terms Clause 1. An antigen binding protein (ABP), comprising a first antigen binding moiety that specifically binds to the internalization domain of a cation-independent mannose-6-phosphate receptor (CI-M6PR), wherein the first antigen binding moiety facilitates intracellular trafficking of the ABP.

[0290] Clause 2. The ABP of clause 1, further comprising a target binding moiety that specifically binds to a target molecule.

[0291] Clause 3. The ABP of Clause 2, wherein said target binding moiety is a second antigen binding moiety that specifically binds to said target molecule.

[0292] Clause 4. The ABP of clause 2, wherein said target binding moiety is attached to said first antigen binding moiety, optionally via a linker.

[0293] Clause 5. The ABP of any one of clauses 2 to 4, wherein said target molecule is a soluble extracellular target molecule or a cell surface target molecule.

[0294] Clause 6. The ABP of any one of the preceding clauses, further comprising a linked cargo moiety.

[0295] Clause 7. The ABP of clause 6, wherein said cargo moiety is a polypeptide fused to said first or second antigen-binding moiety.

[0296] Clause 8. The ABP of clause 6, wherein said cargo moiety is attached to said first or second antigen-binding moiety, optionally via a linker.

[0297] Clause 9. The ABP of clause 1 or 2, wherein said first antigen-binding portion binds to a domain of CI-M6PR selected from domain 1, domain 4, domain 5, domain 6, domain 7, and domain 8.

[0298] Clause 10. The ABP of clause 1 or 2, wherein said first antigen-binding portion binds to domain 1 of CI-M6PR.

[0299] Clause 11. The ABP of clause 1 or 2, wherein said first antigen-binding portion binds to domain 4 of CI-M6PR.

[0300] Clause 12. The ABP of clause 1 or 2, wherein said first antigen-binding portion binds to domain 5 of CI-M6PR.

[0301] Clause 13. The ABP of clause 1 or 2, wherein said first antigen-binding portion binds to domain 6 of CI-M6PR.

[0302] Clause 14. The ABP of clause 1 or 2, wherein said first antigen-binding portion binds to domain 7 of CI-M6PR.

[0303] Clause 15. The ABP of clause 1 or 2, wherein said first antigen-binding portion binds to domain 8 of CI-M6PR.

[0304] Clause 16. The ABP of any one of the preceding clauses, wherein said first antigen-binding portion binds to said CI-M6PR with high affinity.

[0305] Clause 17. The first antigen-binding portion has a dissociation equilibrium constant (K D 17. The ABP of clause 16, which binds to CI-M6PR at

[0306] Clause 18. The first antigen-binding portion has a K of between about 1 nM and about 500 nM. D 10. The ABP of any one of the preceding clauses, which binds to CI-M6PR at

[0307] Clause 19. The first antigen-binding portion has a K of about 10 nM to about 100 nM. D 19. The ABP according to clause 18, which binds to CI-M6PR at

[0308] Clause 20. The first antigen-binding portion has a K of about 100 nM to about 200 nM. D 19. The ABP according to clause 18, which binds to CI-M6PR at

[0309] Clause 21. The first antigen-binding portion has a K of about 200 nM to about 300 nM. D 19. The ABP according to clause 18, which binds to CI-M6PR at

[0310] Clause 22. The first antigen-binding portion has a K of about 300 nM to about 400 nM. D 19. The ABP according to clause 18, which binds to CI-M6PR at

[0311] Clause 23. The first antigen-binding portion has a K of about 400 nM to about 500 nM. D 19. The ABP according to clause 18, which binds to CI-M6PR at

[0312] Clause 24. The dissociation rate (K) of the first antigen-binding moiety to CI-M6PR off ) but 1×10 -8 s -1 ~0.1s -1 2. The ABP of any one of the preceding clauses, wherein

[0313] Clause 25. The K of said first antigen-binding moiety against CI-M6PR off But 1×10 -6 s -1 ~1×10 -2 s -1 2. The ABP of any one of the preceding clauses, wherein

[0314] Clause 26. The K of said first antigen-binding moiety for CI-M6PR off However, about 1 × 10 -5 s -1 10. The ABP of any one of the preceding clauses, wherein:

[0315] Clause 27. The ABP of any one of the preceding clauses, wherein said binding of said first antigen-binding portion to CI-M6PR is pH dependent.

[0316] Clause 28. The ABP of any one of the preceding clauses, wherein said first antigen-binding moiety is released from CI-M6PR at a pH of 7.4 or below.

[0317] Clause 29. The ABP of any one of the preceding clauses, wherein said first antigen-binding portion is released from CI-M6PR at a pH of 6.5 or below.

[0318] Clause 30. The ABP of any one of the preceding clauses, wherein said first antigen-binding moiety is released from CI-M6PR at a pH of 6.0 or below.

[0319] Clause 31. The ABP of any one of the preceding clauses, wherein said first antigen-binding moiety is released from CI-M6PR at a pH of 5.5 or below.

[0320] Clause 32. The ABP of any one of the preceding clauses, wherein said first antigen-binding moiety is released from CI-M6PR at a pH of 5.0 or below.

[0321] Clause 33. The ABP of any one of the preceding clauses, wherein said first antigen-binding portion comprises a light chain CDR3 (LCDR3) having the sequence of SEQ ID NO: 76 and a heavy chain CDR3 (HCDR3) having the sequence of SEQ ID NO: 35.

[0322] Clause 34. The ABP of any one of clauses 1 to 32, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 78 and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 38.

[0323] Clause 35. The ABP of any one of clauses 1 to 32, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 81 and a heavy chain CDR3 having the sequence of SEQ ID NO: 41.

[0324] Clause 36. The ABP of any one of clauses 1 to 32, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 84 and a heavy chain CDR3 having the sequence of SEQ ID NO: 44.

[0325] Clause 37. The ABP of any one of clauses 1 to 32, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 85 and a heavy chain CDR3 having the sequence of SEQ ID NO: 46.

[0326] Clause 38. The ABP of any one of clauses 1 to 32, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 88 and a heavy chain CDR3 having the sequence of SEQ ID NO: 49.

[0327] Clause 39. The ABP of any one of clauses 1 to 32, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 91 and a heavy chain CDR3 having the sequence of SEQ ID NO: 52.

[0328] Clause 40. The ABP of any one of clauses 1 to 32, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 94 and a heavy chain CDR3 having the sequence of SEQ ID NO: 54.

[0329] Clause 41. The ABP of any one of clauses 1 to 32, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 96 and a heavy chain CDR3 having the sequence of SEQ ID NO: 55.

[0330] Clause 42. The ABP of any one of clauses 1 to 32, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 99 and a heavy chain CDR3 having the sequence of SEQ ID NO: 58.

[0331] Clause 43. The ABP of any one of clauses 1 to 32, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 101 and a heavy chain CDR3 having the sequence of SEQ ID NO: 60.

[0332] Clause 44. The ABP of any one of clauses 1 to 32, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 104 and a heavy chain CDR3 having the sequence of SEQ ID NO: 63.

[0333] Clause 45. The ABP of any one of clauses 1 to 32, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 107 and a heavy chain CDR3 having the sequence of SEQ ID NO: 66.

[0334] Clause 46. The ABP of any one of clauses 1 to 32, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 110 and a heavy chain CDR3 having the sequence of SEQ ID NO: 69.

[0335] Clause 47. The ABP of any one of clauses 1 to 32, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 113 and a heavy chain CDR3 having the sequence of SEQ ID NO: 72.

[0336] Clause 48. The ABP of any one of clauses 1 to 32, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 116 and a heavy chain CDR3 having the sequence of SEQ ID NO: 73.

[0337] Clause 49. The ABP of clause 33, wherein the first antigen-binding portion further comprises a light chain CDR1 (LCDR1) having the sequence of SEQ ID NO: 74 and a heavy chain CDR1 (HCDR1) having the sequence of SEQ ID NO: 33, and a light chain CDR2 (LCDR2) having the sequence of SEQ ID NO: 75 and a heavy chain CDR2 (HCDR2) having the sequence of SEQ ID NO: 34.

[0338] Clause 50. The ABP of clause 34, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 36 and a LCDR1 having the sequence of SEQ ID NO: 77, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 37 and a LCDR2 having the sequence of SEQ ID NO: 75.

[0339] Clause 51. The ABP of clause 35, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 39 and a LCDR1 having the sequence of SEQ ID NO: 79, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 80 and a HCDR2 having the sequence of SEQ ID NO: 40.

[0340] Clause 52. The ABP of clause 36, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 42 and a LCDR1 having the sequence of SEQ ID NO: 82, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 43 and a LCDR2 having the sequence of SEQ ID NO: 43.

[0341] Clause 53. The ABP of clause 37, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 79 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 45, and an LCDR2 having the sequence of SEQ ID NO: 80 and a HCDR2 having the sequence of SEQ ID NO: 34.

[0342] Clause 54. The ABP of clause 38, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 86 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 47, and a LCDR2 having the sequence of SEQ ID NO: 87 and a HCDR2 having the sequence of SEQ ID NO: 48.

[0343] Clause 55. The ABP of clause 39, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 89 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 50, and an LCDR2 having the sequence of SEQ ID NO: 90 and a HCDR2 having the sequence of SEQ ID NO: 53.

[0344] Clause 56. The ABP of clause 40, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 92 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 50, and an LCDR2 having the sequence of SEQ ID NO: 93 and a HCDR2 having the sequence of SEQ ID NO: 53.

[0345] Clause 57. The ABP of clause 41, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 95 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 50, and an LCDR2 having the sequence of SEQ ID NO: 93 and a HCDR2 having the sequence of SEQ ID NO: 51.

[0346] Clause 58. The ABP of clause 42, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 97 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 56, and an LCDR2 having the sequence of SEQ ID NO: 98 and a HCDR2 having the sequence of SEQ ID NO: 57.

[0347] Clause 59. The ABP of clause 43, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 100 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 50, and an LCDR2 having the sequence of SEQ ID NO: 93 and a HCDR2 having the sequence of SEQ ID NO: 59.

[0348] Clause 60. The ABP of clause 44, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 102 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 61, and an LCDR2 having the sequence of SEQ ID NO: 103 and a HCDR2 having the sequence of SEQ ID NO: 62.

[0349] Clause 61. The ABP of clause 45, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 105 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 64, and an LCDR2 having the sequence of SEQ ID NO: 106 and a HCDR2 having the sequence of SEQ ID NO: 65.

[0350] Clause 62. The ABP of clause 46, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 108 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 67, and an LCDR2 having the sequence of SEQ ID NO: 109 and a HCDR2 having the sequence of SEQ ID NO: 68.

[0351] Clause 63. The ABP of clause 47, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 111 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 70, and a LCDR2 having the sequence of SEQ ID NO: 112 and a HCDR2 having the sequence of SEQ ID NO: 71.

[0352] Clause 64. The ABP of clause 48, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 114 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 70, and an LCDR2 having the sequence of SEQ ID NO: 115 and a HCDR2 having the sequence of SEQ ID NO: 71.

[0353] Clause 65. The ABP of any one of the preceding clauses, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 205, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 206, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 207, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 208.

[0354] Clause 66. The ABP of any one of the preceding clauses, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 209, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 210, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 211, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 212.

[0355] Clause 67. The ABP of any one of the preceding clauses, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 213, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 214, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 215, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 216.

[0356] Clause 68. The ABP of any one of the preceding clauses, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 217, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 218, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 219, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 220.

[0357] Clause 69. The ABP of any one of the preceding clauses, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 221, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 222, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 223, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 224.

[0358] Clause 70. The ABP of any one of the preceding clauses, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 225, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 226, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 227, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 228.

[0359] Clause 71. The ABP of any one of the preceding clauses, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 229, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 230, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 231, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 232.

[0360] Clause 72. The ABP of any one of the preceding clauses, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 233, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 234, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 235, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 236.

[0361] Clause 73. The ABP of any one of the preceding clauses, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 237, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 238, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 239, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 240.

[0362] Clause 74. The ABP of any one of the preceding clauses, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 241, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 242, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 243, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 244.

[0363] Clause 75. The ABP of any one of the preceding clauses, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 245, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 246, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 247, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 248.

[0364] Clause 76. The ABP of any one of the preceding clauses, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 249, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 250, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 251, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 252.

[0365] Clause 77. The ABP of any one of the preceding clauses, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 253, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 254, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 255, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 256.

[0366] Clause 78. The ABP of any one of the preceding clauses, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 257, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 258, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 259, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 260.

[0367] Clause 79. The ABP of any one of the preceding clauses, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 261, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 262, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 263, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 264.

[0368] Clause 80. The ABP of any one of the preceding clauses, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 265, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 266, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 267, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 268.

[0369] Clause 81. The ABP of clause 65, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 141, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 142, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 143, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 144.

[0370] Clause 82. The ABP of clause 66, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 145, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 146, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 147, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 148.

[0371] Clause 83. The ABP of clause 67, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 149, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 150, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 151, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 152.

[0372] Clause 84. The ABP of clause 68, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 153, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 154, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 155, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 156.

[0373] Clause 85. The ABP of clause 69, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 157, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 158, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 159, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 160.

[0374] Clause 86. The ABP of clause 70, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 161, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 162, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 163, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 164.

[0375] Clause 87. The ABP of clause 71, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 165, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 166, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 167, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 168.

[0376] Clause 88. The ABP of clause 72, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 169, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 170, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 171, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 172.

[0377] Clause 89. The ABP of clause 73, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 173, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 174, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 175, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 176.

[0378] Clause 90. The ABP of clause 74, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 177, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 178, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 179, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 180.

[0379] Clause 91. The ABP of clause 75, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 181, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 182, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 183, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 184.

[0380] Clause 92. The ABP of clause 76, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 185, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 186, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 187, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 188.

[0381] Clause 93. The ABP of clause 77, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 189, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 190, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 191, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 192.

[0382] Clause 94. The ABP of clause 78, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 193, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 194, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 195, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 196.

[0383] Clause 95. The ABP of clause 79, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 197, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 198, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 199, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 200.

[0384] Clause 96. The ABP of clause 80, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 201, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 202, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 203, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 204.

[0385] Clause 97. The ABP of any one of the preceding clauses, wherein the first antigen-binding portion comprises a variable light chain (VL) having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 17-32.

[0386] Clause 98. The ABP of any one of the preceding clauses, wherein the first antigen-binding portion comprises a variable heavy chain (VH) having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 1-16.

[0387] Clause 99. The ABP of any one of the preceding clauses, wherein said first antigen-binding portion is an antibody fragment (e.g., a single-chain variable fragment (scFv) or an antigen-binding fragment).

[0388] Clause 100. The ABP of any one of the preceding clauses, wherein said ABP is transported to a lysosome.

[0389] Clause 101. The ABP of any one of the preceding clauses, wherein said ABP is returned to the cell surface after internalization.

[0390] Clause 102. The ABP of any one of clauses 1 to 100, wherein said ABP is degraded intracellularly.

[0391] Clause 103. An antigen binding protein (ABP) comprising a first antigen binding portion that specifically binds to the cation-independent mannose-6-phosphate receptor (CI-M6PR), comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), a light chain CDR3 (LCDR3), a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), wherein: the LCDR1 has the sequence of SEQ ID NO: 82; the LCDR2 has the sequence of SEQ ID NO: 83, the LCDR3 has the sequence of SEQ ID NO: 84, wherein the HCDR1 has the sequence of SEQ ID NO: 42; the HCDR2 has the sequence of SEQ ID NO: 43, An antigen binding protein (ABP), wherein the HCDR3 has the sequence of SEQ ID NO: 44.

[0392] Clause 104. An antigen binding protein (ABP) comprising a first antigen binding portion that specifically binds to the cation-independent mannose-6-phosphate receptor (CI-M6PR), comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), a light chain CDR3 (LCDR3), a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), wherein: the LCDR1 has the sequence of SEQ ID NO: 92; the LCDR2 has the sequence of SEQ ID NO: 93, the LCDR3 has the sequence of SEQ ID NO: 94, wherein the HCDR1 has the sequence of SEQ ID NO: 50; the HCDR2 has the sequence of SEQ ID NO: 53, An antigen binding protein (ABP), wherein the HCDR3 has the sequence of SEQ ID NO: 54.

[0393] Clause 105. An antigen binding protein (ABP) comprising a first antigen binding portion that specifically binds to the cation-independent mannose-6-phosphate receptor (CI-M6PR), comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), a light chain CDR3 (LCDR3), a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), wherein: the LCDR1 has the sequence of SEQ ID NO: 97; the LCDR2 has the sequence of SEQ ID NO: 98; the LCDR3 has the sequence of SEQ ID NO: 99, wherein the HCDR1 has the sequence of SEQ ID NO: 56; wherein the HCDR2 has the sequence of SEQ ID NO: 57; An antigen binding protein (ABP), wherein the HCDR3 has the sequence of SEQ ID NO: 58.

[0394] Clause 106. An antigen binding protein (ABP) comprising a first antigen binding portion that specifically binds to the cation-independent mannose-6-phosphate receptor (CI-M6PR), comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), a light chain CDR3 (LCDR3), a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), wherein: wherein the LCDR1 has the sequence of SEQ ID NO: 100; wherein the LCDR2 has the sequence of SEQ ID NO: 93; wherein the LCDR3 has the sequence of SEQ ID NO: 101; wherein the HCDR1 has the sequence of SEQ ID NO: 50; wherein said HCDR2 has the sequence of SEQ ID NO: 59; An antigen binding protein (ABP) wherein the HCDR3 has the sequence of SEQ ID NO: 60.

[0395] Clause 107. An antigen binding protein (ABP) comprising a first antigen binding portion that specifically binds to the cation-independent mannose-6-phosphate receptor (CI-M6PR), comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), a light chain CDR3 (LCDR3), a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), wherein: the LCDR1 has the sequence of SEQ ID NO: 108; the LCDR2 has the sequence of SEQ ID NO: 109, the LCDR3 has the sequence of SEQ ID NO: 110; wherein the HCDR1 has the sequence of SEQ ID NO: 67; wherein the HCDR2 has the sequence of SEQ ID NO: 68; An antigen binding protein (ABP), wherein the HCDR3 has the sequence of SEQ ID NO: 69.

[0396] Clause 108. An antigen binding protein (ABP) comprising a first antigen binding portion that specifically binds to the cation-independent mannose-6-phosphate receptor (CI-M6PR), comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), a light chain CDR3 (LCDR3), a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), wherein: the LCDR1 has the sequence of SEQ ID NO: 111; the LCDR2 has the sequence of SEQ ID NO: 112, the LCDR3 has the sequence of SEQ ID NO: 113, wherein the HCDR1 has the sequence of SEQ ID NO: 70; wherein the HCDR2 has the sequence of SEQ ID NO: 71; An antigen binding protein (ABP), wherein the HCDR3 has the sequence of SEQ ID NO: 72.

[0397] Article 109. A bifunctional molecule, a first moiety that specifically binds to the cation-independent mannose-6-phosphate receptor (CI-M6PR), wherein the first moiety is an antibody or an antibody fragment; and a second portion that specifically binds to a cell surface or extracellular target molecule, wherein the second portion is selected from an antibody, an antigen-binding fragment, a ligand, and a small molecule.

[0398] Clause 110. The bifunctional molecule according to Clause 109, wherein said bifunctional molecule is a polypeptide.

[0399] Clause 111. The bifunctional molecule of Clause 109, wherein said first moiety and said second moiety are covalently linked via a linker.

[0400] Clause 112. The bifunctional molecule of Clause 111, wherein said second moiety is a small molecule.

[0401] Clause 113. The bifunctional molecule of any one of clauses 109 to 112, wherein said first binding moiety comprises a light chain CDR3 (LCDR3) having the sequence of SEQ ID NO: 76 and a heavy chain CDR3 (HCDR3) having the sequence of SEQ ID NO: 35.

[0402] Clause 114. The bifunctional molecule of any one of clauses 109 to 112, wherein said first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 78 and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 38.

[0403] Clause 115. The bifunctional molecule of any one of clauses 109 to 112, wherein said first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 81 and a heavy chain CDR3 having the sequence of SEQ ID NO: 41.

[0404] Clause 116. The bifunctional molecule of any one of clauses 109 to 112, wherein said first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 84 and a heavy chain CDR3 having the sequence of SEQ ID NO: 44.

[0405] Clause 117. The bifunctional molecule of any one of clauses 109 to 112, wherein said first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 85 and a heavy chain CDR3 having the sequence of SEQ ID NO: 46.

[0406] Clause 118. The bifunctional molecule of any one of clauses 109 to 112, wherein said first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 88 and a heavy chain CDR3 having the sequence of SEQ ID NO: 49.

[0407] Clause 119. The bifunctional molecule of any one of clauses 109 to 112, wherein said first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 91 and a heavy chain CDR3 having the sequence of SEQ ID NO: 52.

[0408] Clause 120. The bifunctional molecule of any one of clauses 109 to 112, wherein said first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 94 and a heavy chain CDR3 having the sequence of SEQ ID NO: 54.

[0409] Clause 121. The bifunctional molecule of any one of clauses 109 to 112, wherein said first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 96 and a heavy chain CDR3 having the sequence of SEQ ID NO: 55.

[0410] Clause 122. The bifunctional molecule of any one of clauses 109 to 112, wherein said first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 99 and a heavy chain CDR3 having the sequence of SEQ ID NO: 58.

[0411] Clause 123. The bifunctional molecule of any one of clauses 109 to 112, wherein said first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 101 and a heavy chain CDR3 having the sequence of SEQ ID NO: 60.

[0412] Clause 124. The bifunctional molecule of any one of clauses 109 to 112, wherein said first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 104 and a heavy chain CDR3 having the sequence of SEQ ID NO: 63.

[0413] Clause 125. The bifunctional molecule of any one of clauses 109 to 112, wherein said first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 107 and a heavy chain CDR3 having the sequence of SEQ ID NO: 66.

[0414] Clause 126. The bifunctional molecule of any one of clauses 109 to 112, wherein said first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 110 and a heavy chain CDR3 having the sequence of SEQ ID NO: 69.

[0415] Clause 127. The bifunctional molecule of any one of clauses 109 to 112, wherein said first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 113 and a heavy chain CDR3 having the sequence of SEQ ID NO: 72.

[0416] Clause 128. The bifunctional molecule of any one of clauses 109 to 112, wherein said first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 116 and a heavy chain CDR3 having the sequence of SEQ ID NO: 73.

[0417] Clause 129. The bifunctional molecule according to clause 113, wherein the first portion further comprises a light chain CDR1 (LCDR1) having the sequence of SEQ ID NO: 74 and a heavy chain CDR1 (HCDR1) having the sequence of SEQ ID NO: 33, and a light chain CDR2 (LCDR2) having the sequence of SEQ ID NO: 75 and a heavy chain CDR2 (HCDR2) having the sequence of SEQ ID NO: 34.

[0418] Clause 130. The bifunctional molecule according to clause 114, wherein said first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 36 and a LCDR1 having the sequence of SEQ ID NO: 77, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 37 and a LCDR2 having the sequence of SEQ ID NO: 75.

[0419] Clause 131. The bifunctional molecule according to clause 115, wherein said first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 39 and a LCDR1 having the sequence of SEQ ID NO: 79, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 80 and a HCDR2 having the sequence of SEQ ID NO: 40.

[0420] Clause 132. The bifunctional molecule according to clause 116, wherein said first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 42 and a LCDR1 having the sequence of SEQ ID NO: 82, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 43 and a LCDR2 having the sequence of SEQ ID NO: 43.

[0421] Clause 133. The bifunctional molecule according to clause 117, wherein said first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 45 and a LCDR1 having the sequence of SEQ ID NO: 46, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 47 and a heavy chain HCDR2 having the sequence of SEQ ID NO: 48.

[0422] Clause 134. The bifunctional molecule according to clause 118, wherein said first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 86 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 47, and a LCDR2 having the sequence of SEQ ID NO: 87 and a heavy chain HCDR2 having the sequence of SEQ ID NO: 48.

[0423] Clause 135. The bifunctional molecule according to clause 119, wherein said first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 89 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 50, and a LCDR2 having the sequence of SEQ ID NO: 90 and a heavy chain HCDR2 having the sequence of SEQ ID NO: 53.

[0424] Clause 136. The bifunctional molecule according to clause 120, wherein said first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 92 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 50, and a LCDR2 having the sequence of SEQ ID NO: 93 and a heavy chain HCDR2 having the sequence of SEQ ID NO: 53.

[0425] Clause 137. The bifunctional molecule according to clause 121, wherein said first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 95 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 50, and a LCDR2 having the sequence of SEQ ID NO: 93 and a HCDR2 having the sequence of SEQ ID NO: 51.

[0426] Clause 138. The bifunctional molecule according to clause 122, wherein said first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 97 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 56, and a LCDR2 having the sequence of SEQ ID NO: 98 and a heavy chain HCDR2 having the sequence of SEQ ID NO: 57.

[0427] Clause 139. The bifunctional molecule according to clause 123, wherein said first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 100 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 50, and a LCDR2 having the sequence of SEQ ID NO: 93 and a HCDR2 having the sequence of SEQ ID NO: 59.

[0428] Clause 140. The bifunctional molecule according to clause 124, wherein said first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 102 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 61, and a LCDR2 having the sequence of SEQ ID NO: 103 and a HCDR2 having the sequence of SEQ ID NO: 62.

[0429] Clause 141. The bifunctional molecule according to clause 125, wherein said first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 105 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 64, and a LCDR2 having the sequence of SEQ ID NO: 106 and a HCDR2 having the sequence of SEQ ID NO: 65.

[0430] Clause 142. The bifunctional molecule according to clause 126, wherein said first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 108 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 67, and a LCDR2 having the sequence of SEQ ID NO: 109 and a HCDR2 having the sequence of SEQ ID NO: 68.

[0431] Clause 143. The bifunctional molecule according to clause 127, wherein said first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 111 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 70, and a LCDR2 having the sequence of SEQ ID NO: 112 and a HCDR2 having the sequence of SEQ ID NO: 71.

[0432] Clause 144. The bifunctional molecule according to clause 128, wherein said first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 114 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 70, and a LCDR2 having the sequence of SEQ ID NO: 115 and a HCDR2 having the sequence of SEQ ID NO: 71.

[0433] Clause 145. The bifunctional molecule of any one of clauses 109 to 144, wherein said first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 205, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 206, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 207, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 208.

[0434] Clause 146. The bifunctional molecule of any one of clauses 109 to 144, wherein said first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 209, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 210, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 211, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 212.

[0435] Clause 147. The bifunctional molecule of any one of clauses 109 to 144, wherein said first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 213, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 214, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 215, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 216.

[0436] Clause 148. The bifunctional molecule of any one of clauses 109 to 144, wherein said first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 217, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 218, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 219, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 220.

[0437] Clause 149. The bifunctional molecule of any one of clauses 109 to 144, wherein said first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 221, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 222, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 223, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 224.

[0438] Clause 150. The bifunctional molecule of any one of clauses 109 to 144, wherein said first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 225, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 226, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 227, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 228.

[0439] Clause 151. The bifunctional molecule of any one of clauses 109 to 144, wherein said first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 229, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 230, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 231, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 232.

[0440] Clause 152. The bifunctional molecule of any one of clauses 109 to 144, wherein said first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 233, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 234, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 235, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 236.

[0441] Clause 153. The bifunctional molecule of any one of clauses 109 to 144, wherein said first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 237, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 238, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 239, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 240.

[0442] Clause 154. The bifunctional molecule of any one of clauses 109 to 144, wherein said first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 241, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 242, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 243, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 244.

[0443] Clause 155. The bifunctional molecule of any one of clauses 109 to 144, wherein said first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 245, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 246, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 247, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 248.

[0444] Clause 156. The bifunctional molecule of any one of clauses 109 to 144, wherein said first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 249, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 250, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 251, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 252.

[0445] Clause 157. The bifunctional molecule of any one of clauses 109 to 144, wherein said first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 253, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 254, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 255, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 256.

[0446] Clause 158. The bifunctional molecule of any one of clauses 109 to 144, wherein said first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 257, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 258, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 259, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 260.

[0447] Clause 159. The bifunctional molecule of any one of clauses 109 to 144, wherein said first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 261, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 262, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 263, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 264.

[0448] Clause 160. The bifunctional molecule of any one of clauses 109 to 144, wherein said first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 265, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 266, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 267, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 268.

[0449] Clause 161. The bifunctional molecule according to clause 145, wherein said first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 141, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 142, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 143, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 144.

[0450] Clause 162. The bifunctional molecule according to clause 146, wherein said first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 145, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 146, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 147, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 148.

[0451] Clause 163. The bifunctional molecule according to clause 147, wherein said first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 149, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 150, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 151, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 152.

[0452] Clause 164. The bifunctional molecule according to clause 148, wherein said first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 153, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 154, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 155, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 156.

[0453] Clause 165. The bifunctional molecule according to clause 149, wherein said first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 157, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 158, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 159, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 160.

[0454] Clause 166. The bifunctional molecule according to clause 150, wherein said first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 161, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 162, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 163, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 164.

[0455] Clause 167. The bifunctional molecule according to clause 151, wherein said first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 165, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 166, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 167, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 168.

[0456] Clause 168. The bifunctional molecule according to clause 152, wherein said first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 169, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 170, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 171, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 172.

[0457] Clause 169. The bifunctional molecule according to clause 153, wherein said first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 173, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 174, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 175, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 176.

[0458] Clause 170. The bifunctional molecule according to clause 154, wherein said first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 177, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 178, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 179, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 180.

[0459] Clause 171. The bifunctional molecule according to clause 155, wherein said first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 181, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 182, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 183, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 184.

[0460] Clause 172. The bifunctional molecule according to clause 156, wherein said first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 185, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 186, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 187, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 188.

[0461] Clause 173. The bifunctional molecule according to clause 157, wherein said first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 189, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 190, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 191, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 192.

[0462] Clause 174. The bifunctional molecule according to clause 158, wherein said first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 193, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 194, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 195, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 196.

[0463] Clause 175. The bifunctional molecule according to clause 159, wherein said first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 197, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 198, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 199, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 200.

[0464] Clause 176. The bifunctional molecule according to clause 160, wherein said first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 201, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 202, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 203, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 204.

[0465] Clause 177. The bifunctional molecule of any one of clauses 109-176, wherein said first portion comprises a variable light chain (VL) having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 17-32.

[0466] Clause 178. The bifunctional molecule of any one of clauses 109-177, wherein said first portion comprises a variable heavy chain (VH) having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 1-16.

[0467] Clause 179. A method for degrading a soluble or cell surface target molecule, comprising: (a) contacting the target molecule with a multispecific antigen-binding protein (ABP), wherein the ABP comprises a first antigen-binding moiety that specifically binds to a cation-independent mannose-6-phosphate receptor (CI-M6PR) on the surface of a cell; (b) transporting the ABP, the target molecule, and the CI-M6PR to a lysosome within the cell, wherein the target molecule is degraded within the lysosome.

[0468] Clause 180. The method of Clause 179, wherein said ABP further comprises a linked cargo moiety.

[0469] Clause 181. The method of Clause 180, wherein said cargo moiety is a polypeptide fused to said ABP.

[0470] Clause 182. The method of Clause 181, wherein said cargo moiety is attached to said ABP, optionally via a linker.

[0471] Clause 183. The method of any one of clauses 179 to 182, wherein said first antigen-binding portion binds to a domain of CI-M6PR selected from domain 1, domain 4, domain 5, domain 6, domain 7, and domain 8.

[0472] Clause 184. The first antigen-binding portion has a dissociation equilibrium constant (K D 183. The method of any one of clauses 179 to 182, wherein the CI-M6PR is bound by

[0473] Clause 185. The first antigen-binding portion has a dissociation equilibrium constant (K D 183. The method of any one of clauses 179 to 182, wherein the CI-M6PR is bound by

[0474] Clause 186. The dissociation rate (K) of the first antigen-binding moiety for CI-M6PR off ) but 1×10 -8 s -1 ~0.1s -1 186. The method of any one of clauses 179 to 185, wherein

[0475] Clause 187. The method of any one of clauses 179 to 186, wherein said binding of said first antigen-binding portion to CI-M6PR is pH dependent.

[0476] Clause 188. The method of any one of clauses 179 to 187, wherein said first antigen-binding portion is released from CI-M6PR at a pH of 7.4 or below.

[0477] Clause 189. The method of any one of clauses 179 to 188, wherein said first antigen-binding portion is released from CI-M6PR at a pH of 6.0 or below.

[0478] Clause 190. The method of any one of clauses 179 to 189, wherein said first antigen-binding portion is released from CI-M6PR at a pH of 5.5 or below.

[0479] Clause 191. The method of any one of clauses 179-190, wherein said first antigen-binding portion is released from CI-M6PR at a pH of 5.0 or below.

[0480] Clause 192. The method of any one of clauses 179 to 191, wherein said first antigen-binding portion comprises a light chain CDR3 (LCDR3) having the sequence of SEQ ID NO: 76 and a heavy chain CDR3 (HCDR3) having the sequence of SEQ ID NO: 35.

[0481] Clause 193. The method of any one of clauses 179 to 191, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 78 and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 38.

[0482] Clause 194. The method of any one of clauses 179 to 191, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 81 and a heavy chain CDR3 having the sequence of SEQ ID NO: 41.

[0483] Clause 195. The method of any one of clauses 179 to 191, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 84 and a heavy chain CDR3 having the sequence of SEQ ID NO: 44.

[0484] Clause 196. The method of any one of clauses 179 to 191, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 85 and a heavy chain CDR3 having the sequence of SEQ ID NO: 46.

[0485] Clause 197. The method of any one of clauses 179 to 191, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 88 and a heavy chain CDR3 having the sequence of SEQ ID NO: 49.

[0486] Clause 198. The method of any one of clauses 179 to 191, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 91 and a heavy chain CDR3 having the sequence of SEQ ID NO: 52.

[0487] Clause 199. The method of any one of clauses 179 to 191, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 94 and a heavy chain CDR3 having the sequence of SEQ ID NO: 54.

[0488] Clause 200. The method of any one of clauses 179 to 191, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 96 and a heavy chain CDR3 having the sequence of SEQ ID NO: 55.

[0489] Clause 201. The method of any one of clauses 179 to 191, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 99 and a heavy chain CDR3 having the sequence of SEQ ID NO: 58.

[0490] Clause 202. The method of any one of clauses 179 to 191, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 101 and a heavy chain CDR3 having the sequence of SEQ ID NO: 60.

[0491] Clause 203. The method of any one of clauses 179 to 191, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 104 and a heavy chain CDR3 having the sequence of SEQ ID NO: 63.

[0492] Clause 204. The method of any one of clauses 179 to 191, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 107 and a heavy chain CDR3 having the sequence of SEQ ID NO: 66.

[0493] Clause 205. The method of any one of clauses 179 to 191, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 110 and a heavy chain CDR3 having the sequence of SEQ ID NO: 69.

[0494] Clause 206. The method of any one of clauses 179 to 191, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 113 and a heavy chain CDR3 having the sequence of SEQ ID NO: 72.

[0495] Clause 207. The method of any one of clauses 179 to 191, wherein said first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 116 and a heavy chain CDR3 having the sequence of SEQ ID NO: 73.

[0496] Clause 208. The method of clause 192, wherein the first antigen-binding portion further comprises a light chain CDR1 (LCDR1) having the sequence of SEQ ID NO: 74 and a heavy chain CDR1 (HCDR1) having the sequence of SEQ ID NO: 33, and a light chain CDR2 (LCDR2) having the sequence of SEQ ID NO: 75 and a heavy chain CDR2 (HCDR2) having the sequence of SEQ ID NO: 34.

[0497] Clause 209. The method of clause 193, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 77 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 36, and a LCDR2 having the sequence of SEQ ID NO: 75 and a HCDR2 having the sequence of SEQ ID NO: 37.

[0498] Clause 210. The method of clause 194, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 39 and a LCDR1 having the sequence of SEQ ID NO: 79, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 80 and a HCDR2 having the sequence of SEQ ID NO: 40.

[0499] Clause 211. The method of clause 195, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 42 and a LCDR1 having the sequence of SEQ ID NO: 82, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 43 and a LCDR2 having the sequence of SEQ ID NO: 43.

[0500] Clause 212. The method of clause 196, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 79 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 45, and a LCDR2 having the sequence of SEQ ID NO: 80 and a HCDR2 having the sequence of SEQ ID NO: 34.

[0501] Clause 213. The method of clause 197, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 86 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 47, and a LCDR2 having the sequence of SEQ ID NO: 87 and a HCDR2 having the sequence of SEQ ID NO: 48.

[0502] Clause 214. The method of clause 198, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 89 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 50, and a LCDR2 having the sequence of SEQ ID NO: 90 and a HCDR2 having the sequence of SEQ ID NO: 53.

[0503] Clause 215. The method of clause 199, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 92 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 50, and a LCDR2 having the sequence of SEQ ID NO: 93 and a HCDR2 having the sequence of SEQ ID NO: 53.

[0504] Clause 216. The method of clause 200, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 95 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 50, and a LCDR2 having the sequence of SEQ ID NO: 93 and a HCDR2 having the sequence of SEQ ID NO: 51.

[0505] Clause 217. The method of clause 201, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 97 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 56, and a LCDR2 having the sequence of SEQ ID NO: 98 and a HCDR2 having the sequence of SEQ ID NO: 57.

[0506] Clause 218. The method of clause 202, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 100 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 50, and a LCDR2 having the sequence of SEQ ID NO: 93 and a HCDR2 having the sequence of SEQ ID NO: 59.

[0507] Clause 219. The method of clause 203, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 102 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 61, and a LCDR2 having the sequence of SEQ ID NO: 103 and a HCDR2 having the sequence of SEQ ID NO: 62.

[0508] Clause 220. The method of clause 204, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 105 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 64, and a LCDR2 having the sequence of SEQ ID NO: 106 and a HCDR2 having the sequence of SEQ ID NO: 65.

[0509] Clause 221. The method of clause 205, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 108 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 67, and a LCDR2 having the sequence of SEQ ID NO: 109 and a HCDR2 having the sequence of SEQ ID NO: 68.

[0510] Clause 222. The method of clause 206, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 111 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 70, and a LCDR2 having the sequence of SEQ ID NO: 112 and a HCDR2 having the sequence of SEQ ID NO: 71.

[0511] Clause 223. The method of clause 207, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 114 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 70, and a LCDR2 having the sequence of SEQ ID NO: 115 and a HCDR2 having the sequence of SEQ ID NO: 71.

[0512] Clause 224. The method of any one of clauses 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 205, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 206, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 207, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 208.

[0513] Clause 225. The method of any one of clauses 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 209, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 210, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 211, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 212.

[0514] Clause 226. The method of any one of clauses 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 213, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 214, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 215, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 216.

[0515] Clause 227. The method of any one of clauses 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 217, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 218, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 219, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 220.

[0516] Clause 228. The method of any one of clauses 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 221, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 222, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 223, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 224.

[0517] Clause 229. The method of any one of clauses 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 225, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 226, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 227, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 228.

[0518] Clause 230. The method of any one of clauses 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 229, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 230, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 231, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 232.

[0519] Clause 231. The method of any one of clauses 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 233, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 234, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 235, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 236.

[0520] Clause 232. The method of any one of clauses 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 237, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 238, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 239, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 240.

[0521] Clause 233. The method of any one of clauses 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 241, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 242, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 243, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 244.

[0522] Clause 234. The method of any one of clauses 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 245, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 246, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 247, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 248.

[0523] Clause 235. The method of any one of clauses 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 249, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 250, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 251, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 252.

[0524] Clause 236. The method of any one of clauses 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 253, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 254, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 255, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 256.

[0525] Clause 237. The method of any one of clauses 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 257, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 258, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 259, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 260.

[0526] Clause 238. The method of any one of clauses 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 261, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 262, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 263, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 264.

[0527] Clause 239. The method of any one of clauses 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 265, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 266, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 267, and LFR4 is at least 95% identical to the sequence of SEQ ID NO: 268.

[0528] Clause 240. The method of clause 224, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 141, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 142, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 143, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 144.

[0529] Clause 241. The method of clause 225, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 145, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 146, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 147, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 148.

[0530] Clause 242. The method of clause 226, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 149, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 150, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 151, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 152.

[0531] Clause 243. The method of clause 227, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 153, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 154, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 155, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 156.

[0532] Clause 244. The method of clause 228, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 157, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 158, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 159, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 160.

[0533] Clause 245. The method of clause 229, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 161, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 162, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 163, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 164.

[0534] Clause 246. The method of clause 230, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 165, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 166, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 167, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 168.

[0535] Clause 247. The method of clause 231, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 169, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 170, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 171, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 172.

[0536] Clause 248. The method of clause 232, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 173, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 174, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 175, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 176.

[0537] Clause 249. The method of clause 233, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 177, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 178, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 179, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 180.

[0538] Clause 250. The method of clause 234, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 181, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 182, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 183, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 184.

[0539] Clause 251. The method of clause 235, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 185, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 186, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 187, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 188.

[0540] Clause 252. The method of clause 236, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 189, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 190, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 191, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 192.

[0541] Clause 253. The method of clause 237, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 193, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 194, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 195, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 196.

[0542] Clause 254. The method of clause 238, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 197, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 198, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 199, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 200.

[0543] Clause 255. The method of clause 239, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 201, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 202, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 203, and HFR4 is at least 95% identical to the sequence of SEQ ID NO: 204.

[0544] Clause 256. The method of any one of clauses 179-255, wherein said first antigen-binding portion comprises a variable light chain (VL) having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 17-32.

[0545] Clause 257. The method of any one of clauses 179-256, wherein said first antigen-binding portion comprises a variable heavy chain (VH) having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 1-16.

[0546] Clause 258. The method of any one of clauses 179 to 257, wherein said first antigen-binding moiety is an antibody fragment.

[0547] Article 259. The method of any one of clauses 179 to 257, further comprising (c) transporting said ABP to said cell surface.

[0548] Article 260. A method for internalizing a target molecule, comprising: (a) contacting said target molecule with an antigen binding protein (ABP) according to any one of clauses 1 to 108 or a bifunctional molecule according to any one of clauses 109 to 178; (b) internalizing the ABP into a cell.

[0549] 9. Equivalents While the present invention has been particularly shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the invention.

Claims

1. 1. An antigen binding protein (ABP), comprising a first antigen-binding portion that specifically binds to an internalization domain of a cation-independent mannose-6-phosphate receptor (CI-M6PR), which facilitates intracellular transport of the ABP.

2. The ABP of claim 1 , further comprising a target binding moiety that specifically binds to a target molecule.

3. 3. The ABP of claim 2, wherein the target binding moiety is a second antigen binding moiety that specifically binds to the target molecule.

4. 3. The ABP of claim 2, wherein the target binding moiety is attached to the first antigen binding moiety, optionally via a linker.

5. The ABP of any one of claims 2 to 4, wherein the target molecule is a soluble extracellular target molecule or a cell surface target molecule.

6. 10. The ABP of any one of the preceding claims, further comprising a linked cargo moiety.

7. 7. The ABP of claim 6, wherein the cargo moiety is a polypeptide fused to the first or second antigen-binding moiety.

8. 7. The ABP of claim 6, wherein the cargo moiety is attached to the first or second antigen-binding moiety, optionally via a linker.

9. 3. The ABP of claim 1 or 2, wherein the first antigen-binding portion binds to a domain of CI-M6PR selected from domain 1, domain 4, domain 5, domain 6, domain 7, and domain 8.

10. 3. The ABP of claim 1 or 2, wherein the first antigen-binding portion binds to domain 1 of CI-M6PR.

11. 3. The ABP of claim 1 or 2, wherein the first antigen-binding portion binds to domain 4 of CI-M6PR.

12. 3. The ABP of claim 1 or 2, wherein the first antigen-binding portion binds to domain 5 of CI-M6PR.

13. 3. The ABP of claim 1 or 2, wherein the first antigen-binding portion binds to domain 6 of CI-M6PR.

14. 3. The ABP of claim 1 or 2, wherein the first antigen-binding portion binds to domain 7 of CI-M6PR.

15. 3. The ABP of claim 1 or 2, wherein the first antigen-binding portion binds to domain 8 of CI-M6PR.

16. 2. The ABP of any one of the preceding claims, wherein the first antigen-binding portion binds to the CI-M6PR with high affinity.

17. the first antigen-binding moiety has a dissociation equilibrium constant (K Ｄ 17. The ABP of claim 16, which binds to CI-M6PR at

18. the first antigen-binding portion has a K Ｄ 10. The ABP of any one of the preceding claims, which binds to CI-M6PR at

19. the first antigen-binding portion has a K of about 10 nM to about 100 nM Ｄ The ABP of claim 18, which binds to CI-M6PR at

20. the first antigen-binding portion has a K of about 100 nM to about 200 nM Ｄ The ABP of claim 18, which binds to CI-M6PR at

21. the first antigen-binding portion has a K of about 200 nM to about 300 nM Ｄ The ABP of claim 18, which binds to CI-M6PR at

22. the first antigen-binding portion has a K of about 300 nM to about 400 nM Ｄ The ABP of claim 18, which binds to CI-M6PR at

23. the first antigen-binding portion has a K of about 400 nM to about 500 nM Ｄ The ABP of claim 18, which binds to CI-M6PR at

24. The dissociation rate (K ｏｆｆ ) is 1 x 10 －８ s －１ ~0.1s －１ 10. The ABP of any one of the preceding claims, wherein:

25. the K of the first antigen-binding portion for CI-M6PR ｏｆｆ But 1 x 10 －６ s －１ ~1 x 10 －２ s －１ 10. The ABP of any one of the preceding claims, wherein:

26. the K of the first antigen-binding portion for CI-M6PR ｏｆｆ But about 1 x 10 －５ s －１ 10. The ABP of any one of the preceding claims, wherein:

27. 2. The ABP of any one of the preceding claims, wherein the binding of the first antigen-binding moiety to CI-M6PR is pH dependent.

28. 2. The ABP of any one of the preceding claims, wherein the first antigen-binding moiety is released from CI-M6PR at pH 7.4 or below.

29. 2. The ABP of any one of the preceding claims, wherein the first antigen-binding portion is released from CI-M6PR at pH 6.5 or below.

30. 2. The ABP of any one of the preceding claims, wherein the first antigen-binding portion is released from CI-M6PR at pH 6.0 or below.

31. 2. The ABP of any one of the preceding claims, wherein the first antigen-binding portion is released from CI-M6PR at pH 5.5 or below.

32. 2. The ABP of any one of the preceding claims, wherein the first antigen-binding portion is released from CI-M6PR at pH 5.0 or below.

33. 10. The ABP of any one of the preceding claims, wherein the first antigen-binding portion comprises a light chain CDR3 (LCDR3) having the sequence of SEQ ID NO: 76 and a heavy chain CDR3 (HCDR3) having the sequence of SEQ ID NO:

35.

34. 33. The ABP of any one of claims 1 to 32, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 78 and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

38.

35. 33. The ABP of any one of claims 1 to 32, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 81 and a heavy chain CDR3 having the sequence of SEQ ID NO:

41.

36. 33. The ABP of any one of claims 1 to 32, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 84 and a heavy chain CDR3 having the sequence of SEQ ID NO:

44.

37. 33. The ABP of any one of claims 1 to 32, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 85 and a heavy chain CDR3 having the sequence of SEQ ID NO:

46.

38. 33. The ABP of any one of claims 1 to 32, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 88 and a heavy chain CDR3 having the sequence of SEQ ID NO:

49.

39. 33. The ABP of any one of claims 1 to 32, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 91 and a heavy chain CDR3 having the sequence of SEQ ID NO:

52.

40. 33. The ABP of any one of claims 1 to 32, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO:94 and a heavy chain CDR3 having the sequence of SEQ ID NO:

54.

41. 33. The ABP of any one of claims 1 to 32, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 96 and a heavy chain CDR3 having the sequence of SEQ ID NO:

55.

42. 33. The ABP of any one of claims 1 to 32, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO:99 and a heavy chain CDR3 having the sequence of SEQ ID NO:

58.

43. 33. The ABP of any one of claims 1 to 32, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 101 and a heavy chain CDR3 having the sequence of SEQ ID NO:

60.

44. 33. The ABP of any one of claims 1 to 32, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 104 and a heavy chain CDR3 having the sequence of SEQ ID NO:

63.

45. 33. The ABP of any one of claims 1 to 32, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 107 and a heavy chain CDR3 having the sequence of SEQ ID NO:

66.

46. 33. The ABP of any one of claims 1 to 32, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 110 and a heavy chain CDR3 having the sequence of SEQ ID NO:

69.

47. 33. The ABP of any one of claims 1 to 32, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 113 and a heavy chain CDR3 having the sequence of SEQ ID NO:

72.

48. 33. The ABP of any one of claims 1 to 32, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 116 and a heavy chain CDR3 having the sequence of SEQ ID NO:

73.

49. 34. The ABP of claim 33, wherein the first antigen-binding portion further comprises a light chain CDR1 (LCDR1) having the sequence of SEQ ID NO: 74 and a heavy chain CDR1 (HCDR1) having the sequence of SEQ ID NO: 33, and a light chain CDR2 (LCDR2) having the sequence of SEQ ID NO: 75 and a heavy chain CDR2 (HCDR2) having the sequence of SEQ ID NO:

34.

50. 35. The ABP of claim 34, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 36 and a LCDR1 having the sequence of SEQ ID NO: 77, and a LCDR2 having the sequence of SEQ ID NO: 75 and a HCDR2 having the sequence of SEQ ID NO:

37.

51. 36. The ABP of claim 35, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 39 and a LCDR1 having the sequence of SEQ ID NO: 79, and a LCDR2 having the sequence of SEQ ID NO: 80 and a HCDR2 having the sequence of SEQ ID NO:

40.

52. 37. The ABP of claim 36, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 42 and a LCDR1 having the sequence of SEQ ID NO: 82, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 43 and a LCDR2 having the sequence of SEQ ID NO:

83.

53. 38. The ABP of claim 37, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 45 and a LCDR1 having the sequence of SEQ ID NO: 79, and a LCDR2 having the sequence of SEQ ID NO: 80 and a HCDR2 having the sequence of SEQ ID NO:

34.

54. 39. The ABP of claim 38, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 47 and a LCDR1 having the sequence of SEQ ID NO: 86, and a LCDR2 having the sequence of SEQ ID NO: 87 and a HCDR2 having the sequence of SEQ ID NO:

48.

55. 40. The ABP of claim 39, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 50 and a LCDR1 having the sequence of SEQ ID NO: 89, and a LCDR2 having the sequence of SEQ ID NO: 90 and a HCDR2 having the sequence of SEQ ID NO:

53.

56. 41. The ABP of claim 40, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 50 and a LCDR1 having the sequence of SEQ ID NO: 92, and a LCDR2 having the sequence of SEQ ID NO: 93 and a HCDR2 having the sequence of SEQ ID NO:

53.

57. 42. The ABP of claim 41 , wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 50 and a LCDR1 having the sequence of SEQ ID NO: 95, and a LCDR2 having the sequence of SEQ ID NO: 93 and a HCDR2 having the sequence of SEQ ID NO:

51.

58. 43. The ABP of claim 42, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 56 and a LCDR1 having the sequence of SEQ ID NO: 97, and a LCDR2 having the sequence of SEQ ID NO: 98 and a HCDR2 having the sequence of SEQ ID NO:

57.

59. 44. The ABP of claim 43, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 50 and a LCDR1 having the sequence of SEQ ID NO: 100, and a LCDR2 having the sequence of SEQ ID NO: 93 and a HCDR2 having the sequence of SEQ ID NO:

59.

60. 45. The ABP of claim 44, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 61 and a LCDR1 having the sequence of SEQ ID NO: 102, and a LCDR2 having the sequence of SEQ ID NO: 103 and a HCDR2 having the sequence of SEQ ID NO:

62.

61. 46. The ABP of claim 45, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 64 and a LCDR1 having the sequence of SEQ ID NO: 105, and a LCDR2 having the sequence of SEQ ID NO: 106 and a HCDR2 having the sequence of SEQ ID NO:

65.

62. 47. The ABP of claim 46, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 67 and a LCDR1 having the sequence of SEQ ID NO: 109 and a HCDR2 having the sequence of SEQ ID NO:

68.

63. 48. The ABP of claim 47, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 111 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 70, and a LCDR2 having the sequence of SEQ ID NO: 112 and a HCDR2 having the sequence of SEQ ID NO:

71.

64. 49. The ABP of claim 48, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 114 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 70, and a LCDR2 having the sequence of SEQ ID NO: 115 and a HCDR2 having the sequence of SEQ ID NO:

71.

65. 20. The ABP of any one of the preceding claims, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 205, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 206, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 207, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

208.

66. 2. The ABP of any one of the preceding claims, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 209, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 210, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 211, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

212.

67. 2. The ABP of any one of the preceding claims, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 213, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 214, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 215, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

216.

68. 20. The ABP of any one of the preceding claims, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 217, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 218, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 219, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

220.

69. 2. The ABP of any one of the preceding claims, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 221, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 222, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 223, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

224.

70. 2. The ABP of any one of the preceding claims, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 225, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 226, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 227, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

228.

71. 2. The ABP of any one of the preceding claims, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 229, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 230, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 231, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

232.

72. 2. The ABP of any one of the preceding claims, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 233, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 234, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 235, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

236.

73. 2. The ABP of any one of the preceding claims, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 237, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 238, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 239, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

240.

74. 2. The ABP of any one of the preceding claims, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 241, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 242, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 243, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

244.

75. 2. The ABP of any one of the preceding claims, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 245, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 246, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 247, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

248.

76. 2. The ABP of any one of the preceding claims, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 249, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 250, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 251, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

252.

77. 2. The ABP of any one of the preceding claims, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 253, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 254, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 255, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

256.

78. 2. The ABP of any one of the preceding claims, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 257, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 258, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 259, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

260.

79. 2. The ABP of any one of the preceding claims, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 261, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 262, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 263, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

264.

80. 2. The ABP of any one of the preceding claims, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 265, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 266, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 267, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

268.

81. 66. The ABP of claim 65, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 141, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 142, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 143, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

144.

82. 67. The ABP of claim 66, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 145, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 146, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 147, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

148.

83. 68. The ABP of claim 67, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 149, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 150, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 151, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

152.

84. 69. The ABP of claim 68, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 153, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 154, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 155, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

156.

85. 70. The ABP of claim 69, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 157, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 158, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 159, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

160.

86. 71. The ABP of claim 70, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 161, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 162, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 163, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

164.

87. 72. The ABP of claim 71, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 165, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 166, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 167, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

168.

88. 73. The ABP of claim 72, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 169, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 170, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 171, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

172.

89. 74. The ABP of claim 73, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 173, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 174, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 175, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

176.

90. 75. The ABP of claim 74, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 177, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 178, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 179, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

180.

91. 76. The ABP of claim 75, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 181, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 182, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 183, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

184.

92. 77. The ABP of claim 76, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 185, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 186, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 187, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

188.

93. 78. The ABP of claim 77, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 189, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 190, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 191, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

192.

94. 79. The ABP of claim 78, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 193, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 194, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 195, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

196.

95. 80. The ABP of claim 79, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 197, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 198, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 199, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

200.

96. 81. The ABP of claim 80, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 201, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 202, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 203, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

204.

97. 10. The ABP of any one of the preceding claims, wherein the first antigen-binding portion comprises a variable light chain (VL) having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 17-32.

98. 10. The ABP of any one of the preceding claims, wherein the first antigen-binding portion comprises a variable heavy chain (VH) having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 1-16.

99. 10. The ABP of any one of the preceding claims, wherein the first antigen-binding portion is an antibody fragment (e.g., a single-chain variable fragment (scFv) or an antigen-binding fragment).

100. 10. The ABP of any one of the preceding claims, wherein the ABP is transported to a lysosome.

101. 10. The ABP of any one of the preceding claims, wherein the ABP is returned to the cell surface after internalization.

102. The ABP of any one of claims 1 to 100, wherein the ABP is degraded intracellularly.

103. 1. An antigen binding protein (ABP) comprising a first antigen binding portion that specifically binds to the cation-independent mannose-6-phosphate receptor (CI-M6PR), the first antigen binding portion comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), a light chain CDR3 (LCDR3), a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), wherein: the LCDR1 has the sequence of SEQ ID NO: 82; the LCDR2 has the sequence of SEQ ID NO: 83; the LCDR3 has the sequence of SEQ ID NO: 84; said HCDR1 having the sequence of SEQ ID NO: 42; said HCDR2 having the sequence of SEQ ID NO: 43 An antigen binding protein (ABP), wherein said HCDR3 has the sequence of SEQ ID NO:

44.

104. 1. An antigen binding protein (ABP) comprising a first antigen binding portion that specifically binds to the cation-independent mannose-6-phosphate receptor (CI-M6PR), the first antigen binding portion comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), a light chain CDR3 (LCDR3), a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), wherein: the LCDR1 has the sequence of SEQ ID NO: 92; the LCDR2 has the sequence of SEQ ID NO: 93; the LCDR3 has the sequence of SEQ ID NO: 94; said HCDR1 having the sequence of SEQ ID NO: 50; said HCDR2 having the sequence of SEQ ID NO: 53 An antigen binding protein (ABP), wherein said HCDR3 has the sequence of SEQ ID NO:

54.

105. 1. An antigen binding protein (ABP) comprising a first antigen binding portion that specifically binds to the cation-independent mannose-6-phosphate receptor (CI-M6PR), the first antigen binding portion comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), a light chain CDR3 (LCDR3), a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), wherein: the LCDR1 has the sequence of SEQ ID NO: 97; the LCDR2 has the sequence of SEQ ID NO: 98; the LCDR3 has the sequence of SEQ ID NO: 99; said HCDR1 having the sequence of SEQ ID NO: 56; said HCDR2 having the sequence of SEQ ID NO: 57 An antigen binding protein (ABP), wherein said HCDR3 has the sequence of SEQ ID NO:

58.

106. 1. An antigen binding protein (ABP) comprising a first antigen binding portion that specifically binds to the cation-independent mannose-6-phosphate receptor (CI-M6PR), the first antigen binding portion comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), a light chain CDR3 (LCDR3), a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), wherein: said LCDR1 having the sequence of SEQ ID NO: 100; the LCDR2 has the sequence of SEQ ID NO: 93; the LCDR3 has the sequence of SEQ ID NO: 101; said HCDR1 having the sequence of SEQ ID NO: 50; said HCDR2 having the sequence of SEQ ID NO: 59; An antigen binding protein (ABP), wherein said HCDR3 has the sequence of SEQ ID NO:

60.

107. 1. An antigen binding protein (ABP) comprising a first antigen binding portion that specifically binds to the cation-independent mannose-6-phosphate receptor (CI-M6PR), the first antigen binding portion comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), a light chain CDR3 (LCDR3), a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), wherein: the LCDR1 has the sequence of SEQ ID NO: 108; the LCDR2 has the sequence of SEQ ID NO: 109; the LCDR3 has the sequence of SEQ ID NO: 110; said HCDR1 having the sequence of SEQ ID NO: 67; said HCDR2 having the sequence of SEQ ID NO: 68 An antigen binding protein (ABP), wherein said HCDR3 has the sequence of SEQ ID NO:

69.

108. 1. An antigen binding protein (ABP) comprising a first antigen binding portion that specifically binds to the cation-independent mannose-6-phosphate receptor (CI-M6PR), the first antigen binding portion comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), a light chain CDR3 (LCDR3), a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), wherein: the LCDR1 has the sequence of SEQ ID NO: 111; the LCDR2 has the sequence of SEQ ID NO: 112; the LCDR3 has the sequence of SEQ ID NO: 113; said HCDR1 having the sequence of SEQ ID NO: 70; said HCDR2 having the sequence of SEQ ID NO: 71 An antigen binding protein (ABP), wherein said HCDR3 has the sequence of SEQ ID NO:

72.

109. A bifunctional molecule comprising: a first moiety that specifically binds to the cation-independent mannose-6-phosphate receptor (CI-M6PR), a first moiety, wherein the first moiety is an antibody or an antibody fragment; a second portion that specifically binds to a cell surface or extracellular target molecule, said second portion being selected from an antibody, an antigen-binding fragment, a ligand, and a small molecule.

110. 110. The bifunctional molecule of claim 109, wherein the bifunctional molecule is a polypeptide.

111. 110. The bifunctional molecule of claim 109, wherein the first portion and the second portion are covalently linked via a linker.

112. 112. The bifunctional molecule of claim 111, wherein the second moiety is a small molecule.

113. 113. The bifunctional molecule of any one of claims 109 to 112, wherein the first binding moiety comprises a light chain CDR3 (LCDR3) having the sequence of SEQ ID NO: 76 and a heavy chain CDR3 (HCDR3) having the sequence of SEQ ID NO:

35.

114. 113. The bifunctional molecule of any one of claims 109 to 112, wherein the first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 78 and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

38.

115. 113. The bifunctional molecule of any one of claims 109 to 112, wherein the first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 81 and a heavy chain CDR3 having the sequence of SEQ ID NO:

41.

116. 113. The bifunctional molecule of any one of claims 109 to 112, wherein the first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 84 and a heavy chain CDR3 having the sequence of SEQ ID NO:

44.

117. 113. The bifunctional molecule of any one of claims 109 to 112, wherein the first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 85 and a heavy chain CDR3 having the sequence of SEQ ID NO:

46.

118. 113. The bifunctional molecule of any one of claims 109 to 112, wherein the first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 88 and a heavy chain CDR3 having the sequence of SEQ ID NO:

49.

119. 113. The bifunctional molecule of any one of claims 109 to 112, wherein the first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 91 and a heavy chain CDR3 having the sequence of SEQ ID NO:

52.

120. 113. The bifunctional molecule of any one of claims 109 to 112, wherein the first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 94 and a heavy chain CDR3 having the sequence of SEQ ID NO:

54.

121. 113. The bifunctional molecule of any one of claims 109 to 112, wherein the first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 96 and a heavy chain CDR3 having the sequence of SEQ ID NO:

55.

122. 113. The bifunctional molecule of any one of claims 109 to 112, wherein the first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 99 and a heavy chain CDR3 having the sequence of SEQ ID NO:

58.

123. 113. The bifunctional molecule of any one of claims 109 to 112, wherein the first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 101 and a heavy chain CDR3 having the sequence of SEQ ID NO:

60.

124. 113. The bifunctional molecule of any one of claims 109 to 112, wherein the first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 104 and a heavy chain CDR3 having the sequence of SEQ ID NO:

63.

125. 113. The bifunctional molecule of any one of claims 109 to 112, wherein the first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 107 and a heavy chain CDR3 having the sequence of SEQ ID NO:

66.

126. 113. The bifunctional molecule of any one of claims 109 to 112, wherein the first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 110 and a heavy chain CDR3 having the sequence of SEQ ID NO:

69.

127. 113. The bifunctional molecule of any one of claims 109 to 112, wherein the first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 113 and a heavy chain CDR3 having the sequence of SEQ ID NO:

72.

128. 113. The bifunctional molecule of any one of claims 109 to 112, wherein the first binding moiety comprises a light chain CDR3 having the sequence of SEQ ID NO: 116 and a heavy chain CDR3 having the sequence of SEQ ID NO:

73.

129. The bifunctional molecule of claim 113, wherein the first portion further comprises a light chain CDR1 (LCDR1) having the sequence of SEQ ID NO: 74 and a heavy chain CDR1 (HCDR1) having the sequence of SEQ ID NO: 33, and a light chain CDR2 (LCDR2) having the sequence of SEQ ID NO: 75 and a heavy chain CDR2 (HCDR2) having the sequence of SEQ ID NO:

34.

130. The bifunctional molecule of claim 114, wherein the first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 36 and a LCDR1 having the sequence of SEQ ID NO: 77, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 36 and a LCDR2 having the sequence of SEQ ID NO: 75 and a HCDR2 having the sequence of SEQ ID NO:

37.

131. The bifunctional molecule of claim 115, wherein the first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 39 and a LCDR1 having the sequence of SEQ ID NO: 79, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 80 and a HCDR2 having the sequence of SEQ ID NO:

40.

132. The bifunctional molecule of claim 116, wherein the first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 42 and a LCDR1 having the sequence of SEQ ID NO: 82, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 43 and a LCDR2 having the sequence of SEQ ID NO:

83.

133. The bifunctional molecule of claim 117, wherein the first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 45 and a LCDR1 having the sequence of SEQ ID NO: 79, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 80 and a HCDR2 having the sequence of SEQ ID NO:

34.

134. The bifunctional molecule of claim 118, wherein the first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 47 and a LCDR1 having the sequence of SEQ ID NO: 86, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 47 and a LCDR2 having the sequence of SEQ ID NO: 87 and a HCDR2 having the sequence of SEQ ID NO:

48.

135. The bifunctional molecule of claim 119, wherein the first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 50 and a LCDR1 having the sequence of SEQ ID NO: 89, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 90 and a HCDR2 having the sequence of SEQ ID NO:

53.

136. The bifunctional molecule of claim 120, wherein the first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 50 and a LCDR1 having the sequence of SEQ ID NO: 92, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 53 and a LCDR2 having the sequence of SEQ ID NO:

93.

137. The bifunctional molecule of claim 121, wherein the first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 50 and a LCDR1 having the sequence of SEQ ID NO: 95, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 51 and a LCDR2 having the sequence of SEQ ID NO:

93.

138. The bifunctional molecule of claim 122, wherein the first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 56 and a LCDR1 having the sequence of SEQ ID NO: 97, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 56 and a LCDR2 having the sequence of SEQ ID NO:

98.

139. The bifunctional molecule of claim 123, wherein the first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 50 and a LCDR1 having the sequence of SEQ ID NO: 100, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 93 and a HCDR2 having the sequence of SEQ ID NO:

59.

140. The bifunctional molecule of claim 124, wherein the first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 61 and a LCDR1 having the sequence of SEQ ID NO: 102, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 62 and a LCDR2 having the sequence of SEQ ID NO:

103.

141. The bifunctional molecule of claim 125, wherein the first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 64 and a LCDR1 having the sequence of SEQ ID NO: 105, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 106 and a HCDR2 having the sequence of SEQ ID NO:

65.

142. The bifunctional molecule of claim 126, wherein the first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 67 and a LCDR1 having the sequence of SEQ ID NO: 109 and a HCDR2 having the sequence of SEQ ID NO:

68.

143. The bifunctional molecule of claim 127, wherein the first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 70 and a LCDR1 having the sequence of SEQ ID NO: 111, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 112 and a HCDR2 having the sequence of SEQ ID NO:

71.

144. The bifunctional molecule of claim 128, wherein the first portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 114 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 70, and a LCDR2 having the sequence of SEQ ID NO: 115 and a HCDR2 having the sequence of SEQ ID NO:

71.

145. 145. The bifunctional molecule of any one of claims 109 to 144, wherein the first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 205, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 206, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 207, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

208.

146. 145. The bifunctional molecule of any one of claims 109 to 144, wherein the first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 209, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 210, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 211, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

212.

147. 145. The bifunctional molecule of any one of claims 109 to 144, wherein the first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 213, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 214, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 215, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

216.

148. 145. The bifunctional molecule of any one of claims 109 to 144, wherein the first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 217, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 218, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 219, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

220.

149. 145. The bifunctional molecule of any one of claims 109 to 144, wherein the first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 221, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 222, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 223, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

224.

150. 145. The bifunctional molecule of any one of claims 109 to 144, wherein the first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 225, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 226, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 227, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

228.

151. 145. The bifunctional molecule of any one of claims 109 to 144, wherein the first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 229, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 230, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 231, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

232.

152. 145. The bifunctional molecule of any one of claims 109 to 144, wherein the first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 233, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 234, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 235, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

236.

153. 145. The bifunctional molecule of any one of claims 109 to 144, wherein the first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 237, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 238, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 239, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

240.

154. 145. The bifunctional molecule of any one of claims 109 to 144, wherein the first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 241, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 242, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 243, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

244.

155. 145. The bifunctional molecule of any one of claims 109 to 144, wherein the first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 245, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 246, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 247, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

248.

156. 145. The bifunctional molecule of any one of claims 109 to 144, wherein the first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 249, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 250, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 251, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

252.

157. 145. The bifunctional molecule of any one of claims 109 to 144, wherein the first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 253, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 254, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 255, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

256.

158. 145. The bifunctional molecule of any one of claims 109 to 144, wherein the first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 257, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 258, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 259, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

260.

159. 145. The bifunctional molecule of any one of claims 109 to 144, wherein the first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 261, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 262, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 263, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

264.

160. 145. The bifunctional molecule of any one of claims 109 to 144, wherein the first portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 265, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 266, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 267, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

268.

161. The bifunctional molecule of claim 145, wherein the first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 141, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 142, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 143, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

144.

162. The bifunctional molecule of claim 146, wherein the first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 145, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 146, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 147, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

148.

163. The bifunctional molecule of claim 147, wherein the first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 149, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 150, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 151, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

152.

164. The bifunctional molecule of claim 148, wherein the first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 153, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 154, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 155, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

156.

165. The bifunctional molecule of claim 149, wherein the first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 157, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 158, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 159, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

160.

166. The bifunctional molecule of claim 150, wherein the first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 161, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 162, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 163, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

164.

167. The bifunctional molecule of claim 151, wherein the first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 165, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 166, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 167, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

168.

168. The bifunctional molecule of claim 152, wherein the first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 169, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 170, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 171, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

172.

169. The bifunctional molecule of claim 153, wherein the first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 173, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 174, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 175, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

176.

170. The bifunctional molecule of claim 154, wherein the first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 177, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 178, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 179, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

180.

171. The bifunctional molecule of claim 155, wherein the first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 181, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 182, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 183, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

184.

172. The bifunctional molecule of claim 156, wherein the first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 185, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 186, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 187, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

188.

173. The bifunctional molecule of claim 157, wherein the first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 189, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 190, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 191, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

192.

174. The bifunctional molecule of claim 158, wherein the first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 193, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 194, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 195, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

196.

175. The bifunctional molecule of claim 159, wherein the first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 197, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 198, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 199, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

200.

176. The bifunctional molecule of claim 160, wherein the first portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 201, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 202, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 203, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

204.

177. 177. The bifunctional molecule of any one of claims 109-176, wherein the first portion comprises a variable light chain (VL) having an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 17-32.

178. 178. The bifunctional molecule of any one of claims 109-177, wherein the first portion comprises a variable heavy chain (VH) having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 1-16.

179. 1. A method for degrading a soluble or cell surface target molecule, comprising: (a) contacting the target molecule with a multispecific antigen-binding protein (ABP), wherein the ABP comprises a first antigen-binding moiety that specifically binds to a cation-independent mannose-6-phosphate receptor (CI-M6PR) on the surface of a cell; (b) transporting the ABP, the target molecule, and the CI-M6PR to a lysosome within the cell, wherein the target molecule is degraded within the lysosome.

180. 180. The method of claim 179, wherein the ABP further comprises a linked cargo moiety.

181. 181. The method of claim 180, wherein the cargo moiety is a polypeptide fused to the ABP.

182. 182. The method of claim 181, wherein the cargo moiety is attached to the ABP, optionally via a linker.

183. 183. The method of any one of claims 179 to 182, wherein the first antigen-binding portion binds to a domain of CI-M6PR selected from domain 1, domain 4, domain 5, domain 6, domain 7, and domain 8.

184. the first antigen-binding moiety has a dissociation equilibrium constant (K Ｄ The method of any one of claims 179 to 182, wherein the CI-M6PR is bound by

185. the first antigen-binding moiety has a dissociation equilibrium constant (K Ｄ The method of any one of claims 179 to 182, wherein the CI-M6PR is bound by

186. The dissociation rate (k ｏｆｆ ) is 1 x 10 －８ s －１ ~0.1s －１ The method according to any one of claims 179 to 185, wherein

187. 187. The method of any one of claims 179 to 186, wherein the binding of the first antigen-binding moiety to CI-M6PR is pH dependent.

188. 188. The method of any one of claims 179 to 187, wherein the first antigen-binding portion is released from CI-M6PR at pH 7.4 or below.

189. 189. The method of any one of claims 179 to 188, wherein the first antigen-binding portion is released from CI-M6PR at pH 6.0 or below.

190. 190. The method of any one of claims 179 to 189, wherein the first antigen-binding portion is released from CI-M6PR at pH 5.5 or less.

191. 191. The method of any one of claims 179 to 190, wherein the first antigen-binding portion is released from CI-M6PR at pH 5.0 or below.

192. 192. The method of any one of claims 179 to 191, wherein the first antigen-binding portion comprises a light chain CDR3 (LCDR3) having the sequence of SEQ ID NO: 76 and a heavy chain CDR3 (HCDR3) having the sequence of SEQ ID NO:

35.

193. 192. The method of any one of claims 179 to 191, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 78 and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

38.

194. 192. The method of any one of claims 179 to 191, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 81 and a heavy chain CDR3 having the sequence of SEQ ID NO:

41.

195. 192. The method of any one of claims 179 to 191, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 84 and a heavy chain CDR3 having the sequence of SEQ ID NO:

44.

196. 192. The method of any one of claims 179 to 191, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 85 and a heavy chain CDR3 having the sequence of SEQ ID NO:

46.

197. 192. The method of any one of claims 179 to 191, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 88 and a heavy chain CDR3 having the sequence of SEQ ID NO:

49.

198. 192. The method of any one of claims 179 to 191, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 91 and a heavy chain CDR3 having the sequence of SEQ ID NO:

52.

199. 192. The method of any one of claims 179 to 191, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 94 and a heavy chain CDR3 having the sequence of SEQ ID NO:

54.

200. 192. The method of any one of claims 179 to 191, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 96 and a heavy chain CDR3 having the sequence of SEQ ID NO:

55.

201. 192. The method of any one of claims 179 to 191, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 99 and a heavy chain CDR3 having the sequence of SEQ ID NO:

58.

202. 192. The method of any one of claims 179 to 191, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 101 and a heavy chain CDR3 having the sequence of SEQ ID NO:

60.

203. 192. The method of any one of claims 179 to 191, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 104 and a heavy chain CDR3 having the sequence of SEQ ID NO:

63.

204. 192. The method of any one of claims 179 to 191, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 107 and a heavy chain CDR3 having the sequence of SEQ ID NO:

66.

205. 192. The method of any one of claims 179 to 191, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 110 and a heavy chain CDR3 having the sequence of SEQ ID NO:

69.

206. 192. The method of any one of claims 179 to 191, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 113 and a heavy chain CDR3 having the sequence of SEQ ID NO:

72.

207. 192. The method of any one of claims 179 to 191, wherein the first antigen-binding portion comprises a light chain CDR3 having the sequence of SEQ ID NO: 116 and a heavy chain CDR3 having the sequence of SEQ ID NO:

73.

208. 193. The method of claim 192, wherein the first antigen-binding portion further comprises a light chain CDR1 (LCDR1) having the sequence of SEQ ID NO: 74 and a heavy chain CDR1 (HCDR1) having the sequence of SEQ ID NO: 33, and a light chain CDR2 (LCDR2) having the sequence of SEQ ID NO: 75 and a heavy chain CDR2 (HCDR2) having the sequence of SEQ ID NO:

34.

209. The method of claim 193, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 36 and a LCDR1 having the sequence of SEQ ID NO: 77, and a LCDR2 having the sequence of SEQ ID NO: 75 and a HCDR2 having the sequence of SEQ ID NO:

37.

210. 195. The method of claim 194, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 39 and a LCDR1 having the sequence of SEQ ID NO: 79, and a LCDR2 having the sequence of SEQ ID NO: 80 and a HCDR2 having the sequence of SEQ ID NO:

40.

211. 196. The method of claim 195, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 42 and a LCDR1 having the sequence of SEQ ID NO: 82, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 43 and a LCDR2 having the sequence of SEQ ID NO:

43.

212. 200. The method of claim 196, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 45 and a LCDR1 having the sequence of SEQ ID NO: 79, and a LCDR2 having the sequence of SEQ ID NO: 80 and a HCDR2 having the sequence of SEQ ID NO:

34.

213. The method of claim 197, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 47 and a LCDR1 having the sequence of SEQ ID NO: 86, and a LCDR2 having the sequence of SEQ ID NO: 87 and a HCDR2 having the sequence of SEQ ID NO:

48.

214. 200. The method of claim 198, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 50 and a LCDR1 having the sequence of SEQ ID NO: 89, and a LCDR2 having the sequence of SEQ ID NO: 90 and a HCDR2 having the sequence of SEQ ID NO:

53.

215. 200. The method of claim 199, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 50 and a LCDR1 having the sequence of SEQ ID NO: 92, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 53 and a LCDR2 having the sequence of SEQ ID NO:

93.

216. The method of claim 200, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 50 and a LCDR1 having the sequence of SEQ ID NO: 95, and a LCDR2 having the sequence of SEQ ID NO: 93 and a HCDR2 having the sequence of SEQ ID NO:

51.

217. The method of claim 201, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 56 and a LCDR1 having the sequence of SEQ ID NO: 97, and a LCDR2 having the sequence of SEQ ID NO: 98 and a HCDR2 having the sequence of SEQ ID NO:

57.

218. The method of claim 202, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 50 and a LCDR1 having the sequence of SEQ ID NO: 100, and a LCDR2 having the sequence of SEQ ID NO: 93 and a HCDR2 having the sequence of SEQ ID NO:

59.

219. The method of claim 203, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 61 and a LCDR1 having the sequence of SEQ ID NO: 102, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 62 and a LCDR2 having the sequence of SEQ ID NO:

103.

220. The method of claim 204, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 64 and a LCDR1 having the sequence of SEQ ID NO: 105, and a LCDR2 having the sequence of SEQ ID NO: 106 and a HCDR2 having the sequence of SEQ ID NO:

65.

221. The method of claim 205, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 67 and a LCDR1 having the sequence of SEQ ID NO: 109 and a HCDR2 having the sequence of SEQ ID NO:

68.

222. The method of claim 206, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 70 and a LCDR1 having the sequence of SEQ ID NO: 111, and a heavy chain HCDR2 having the sequence of SEQ ID NO: 112 and a HCDR2 having the sequence of SEQ ID NO:

71.

223. The method of claim 207, wherein the first antigen-binding portion further comprises a heavy chain HCDR1 having the sequence of SEQ ID NO: 114 and a heavy chain HCDR1 having the sequence of SEQ ID NO: 70, and a LCDR2 having the sequence of SEQ ID NO: 115 and a HCDR2 having the sequence of SEQ ID NO:

71.

224. 224. The method of any one of claims 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 205, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 206, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 207, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

208.

225. 224. The method of any one of claims 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 209, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 210, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 211, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

212.

226. 224. The method of any one of claims 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 213, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 214, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 215, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

216.

227. 224. The method of any one of claims 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 217, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 218, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 219, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

220.

228. 224. The method of any one of claims 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 221, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 222, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 223, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

224.

229. 224. The method of any one of claims 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 225, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 226, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 227, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

228.

230. 224. The method of any one of claims 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 229, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 230, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 231, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

232.

231. 224. The method of any one of claims 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 233, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 234, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 235, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

236.

232. 224. The method of any one of claims 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 237, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 238, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 239, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

240.

233. 224. The method of any one of claims 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 241, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 242, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 243, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

244.

234. 224. The method of any one of claims 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 245, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 246, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 247, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

248.

235. 224. The method of any one of claims 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 249, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 250, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 251, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

252.

236. 224. The method of any one of claims 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 253, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 254, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 255, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

256.

237. 224. The method of any one of claims 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 257, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 258, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 259, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

260.

238. 224. The method of any one of claims 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 261, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 262, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 263, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

264.

239. 224. The method of any one of claims 179 to 223, wherein the first antigen-binding portion comprises a variable light chain (VL) comprising framework regions LFR1, LFR2, LFR3, and LFR4, wherein LFR1 is at least 95% identical to the sequence of SEQ ID NO: 265, LFR2 is at least 95% identical to the sequence of SEQ ID NO: 266, LFR3 is at least 95% identical to the sequence of SEQ ID NO: 267, and LFR4 is at least 95% identical to the sequence of SEQ ID NO:

268.

240. The first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein: The method of claim 224, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 141, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 142, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 143, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

144.

241. The method of claim 225, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 145, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 146, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 147, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

148.

242. 227. The method of claim 226, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 149, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 150, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 151, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

152.

243. 228. The method of claim 227, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 153, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 154, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 155, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

156.

244. 229. The method of claim 228, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 157, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 158, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 159, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

160.

245. 230. The method of claim 229, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 161, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 162, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 163, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

164.

246. The method of claim 230, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 165, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 166, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 167, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

168.

247. The method of claim 231, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 169, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 170, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 171, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

172.

248. 233. The method of claim 232, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 173, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 174, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 175, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

176.

249. The method of claim 233, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 177, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 178, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 179, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

180.

250. The method of claim 234, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 181, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 182, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 183, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

184.

251. The method of claim 235, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 185, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 186, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 187, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

188.

252. The method of claim 236, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 189, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 190, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 191, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

192.

253. The method of claim 237, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 193, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 194, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 195, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

196.

254. The method of claim 238, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 197, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 198, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 199, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

200.

255. 240. The method of claim 239, wherein the first antigen-binding portion comprises a variable heavy chain (VH) comprising framework regions HFR1, HFR2, HFR3, and HFR4, wherein HFR1 is at least 95% identical to the sequence of SEQ ID NO: 201, HFR2 is at least 95% identical to the sequence of SEQ ID NO: 202, HFR3 is at least 95% identical to the sequence of SEQ ID NO: 203, and HFR4 is at least 95% identical to the sequence of SEQ ID NO:

204.

256. 256. The method of any one of claims 179-255, wherein the first antigen-binding portion comprises a variable light chain (VL) having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 17-32.

257. 257. The method of any one of claims 179-256, wherein the first antigen-binding portion comprises a variable heavy chain (VH) having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 1-16.

258. 258. The method of any one of claims 179 to 257, wherein the first antigen-binding portion is an antibody fragment.

259. 258. The method of any one of claims 179-257, further comprising (c) transporting the ABP to the cell surface.

260. 1. A method for internalizing a target molecule, comprising: (a) contacting the target molecule with an antigen binding protein (ABP) of any one of claims 1 to 108 or a bifunctional molecule of any one of claims 109 to 178; (b) internalizing the ABP into the cell.

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