CN116997356A - Anti-ALPP/ALPPL2 antibodies and antibody-drug conjugates - Google Patents

Abstract

Antigen binding proteins, such as antibodies and fragments thereof, that bind ALPP and/or ALPPL2 are provided. Nucleic acids encoding such antigen-binding proteins and vectors and cells useful for preparing such antigen-binding proteins are also provided. The antigen-binding proteins can be used in a variety of methods, including the treatment of ovarian cancer.

Description

Anti-ALPP/ALPPL2 Antibodies and Antibody-Drug Conjugates

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/162,635, filed on March 18, 2021, and U.S. Provisional Application No. 63/301,574, filed on January 21, 2022, each of which is incorporated by reference in its entirety for all purposes.

References to sequence listings

This application includes an electronic sequence listing in a file named 5620-00112PC_ST25 created on March 4, 2022 and containing 106Kb, which is hereby incorporated by reference.

Technical Field

The present invention relates to novel anti-ALPP/ALPPL2 antibodies and antibody-drug conjugates and methods of using such anti-ALPP/ALPPL2 antibodies and antibody-drug conjugates to treat cancer.

Background Art

ALPP (also known as placental alkaline phosphatase) and ALPPL2 (also known as placental-like alkaline phosphatase 2) are paralogous genes expressed primarily in the placenta. ALPP and ALPPL2 are membrane-bound proteins involved in the recycling of ATP from the extracellular space. ALPP is upregulated in a variety of cancers, including ovarian cancer, lung cancer, endometrial cancer, bladder cancer, and gastric cancer. ALPPL2 is also upregulated in a variety of cancers, including ovarian cancer, lung cancer, endometrial cancer, bladder cancer, gastric cancer, and testicular cancer. Ovarian cancer is the fifth most common gynecological malignancy, and improved treatments for this disease are needed.

All references cited herein, including patent applications, patent publications, and scientific literature, are hereby incorporated by reference in their entirety to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

Summary of the invention

Anti-ALPP antibodies and antibody-drug conjugates (ADCs) for ALPP, and anti-ALPPL2 antibodies and ADCs for ALPPL2 are provided herein. Antibodies that can bind to both ALPP and ALPPL2 (anti-ALPP/ALPPL2 antibodies) and ADCs for both ALPP and ALPPL2 (ADCs for ALPP/ALPPL2) are also provided herein. Methods of using antibodies and ADCs for anti-ALPP/ALPPL2 to treat conditions (including cancer) expressing ALPP and/or ALPPL2 are also provided herein. In some embodiments, the anti-ALPP/ALPPL2 antibody comprises heavy chain CDR sequences of SEQ ID NOs: 56, 57, and 58 and light chain CDR sequences of SEQ ID NOs: 63, 64, and 65, as determined by Kabat numbering. In some embodiments, the anti-ALPP/ALPPL2 antibody comprises heavy chain CDR sequences of SEQ ID NOs: 60, 61, and 62 and light chain CDR sequences of SEQ ID NOs: 66, 67, and 68, as determined by IMGT numbering.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the cytotoxicity evaluation of ALPP/ALPPL2-specific antibodies as ADCs using payloads with and without bystander activity against COV644 and NCI-H1651 tumor cells.

Figures 2A to 2C show the remaining viability of cell lines CAOV3, COV644, and NCI-H1651 after cytotoxicity assessment of top ALPP/ALPPL2-specific antibodies as ADCs using payloads without bystander activity.

FIG3 shows the binding affinity of ALPP/ALPPL2-specific antibodies.

FIG4 shows the complete binding profiles of antibodies 1F7 and 12F3 to HEK293 cells expressing ALPP and ALPPL2 as measured by flow cytometry.

Figure 5 shows the sequence alignment of the h12F3 variable heavy chain variant with the human heavy chain acceptor sequence IGHV3-49/HJ4.

Figure 6 shows the variable domain alignment of h12F3 variable heavy chain variants.

Figure 7 shows the sequence alignment of the h12F3 variable light chain variant with the human kappa receptor sequence IGKV1-33/KJ2.

Figure 8 shows the variable domain alignment of the h12F3 light chain variants.

Fig. 9 shows the percentage of surviving CAOV3 cells. The left side of the figure shows the activity percentage of the dose-response curve of the ADC containing humanized F light chain variants and different heavy chains. The right side of the figure shows the activity of the ADC containing humanized D heavy chain and different light chain variants.

FIG. 10 shows the in vitro potency correlation between humanized variants with the mp-dLAE-MMAE (4) drug linker.

Figure 11 shows the cytotoxicity profile of h12F3 HGLF ADC on 3D spheroids in vitro. Kinetic binding curves and values for h12F3 HGLF and HFLD variants with ALPP and ALPPL2 Fc fusions are shown.

FIG. 12 shows the internalization kinetics of h12F3 HGLF at different antibody concentrations.

Figure 13 shows kinetic binding curves and values for h12F3 HGLF and HFLD variants with ALPP and ALPPL2 Fc fusions.

FIG. 14 shows the kinetic binding curves and values of h12F3 HGLF and HFLD to ALPP and ALPPL2 fc fusions at pH 7.4 and pH 6.

FIG. 15 shows the in vivo anti-tumor activity of h12F3 HGLF-dLAE-MMAE and HFLD-dLAE-MMAE in the CAOV3 mouse model.

FIG. 16 shows the in vivo anti-tumor activity of h12F3 HGLF-dLAE-MMAE and HFLD-dLAE-MMAE in the NCI-N87 mouse model.

FIG. 17 shows the in vivo anti-tumor activity of h12F3 HFLD-dLAE-MMAE and HFLD-vc-MMAE in the NCI-N87 mouse model.

FIG. 18 shows the in vivo anti-tumor activity of h12F3 HFLD-dLAE-MMAE and HFLD-vc-MMAE in the H1651 mouse model.

FIG. 19 shows the % change in tumor volume in seven xenograft models following treatment with h12F3 ADC conjugated to vc-MMAE and dLAE-MMAE.

FIG. 20 shows the in vivo anti-tumor activity of h12F3 HGLF-mc-vc-MMAE or h12F3 HGLF-mp-dL AE-MMAE conjugates compared to their respective isotype ADC controls in the NCI-N87 gastric model.

FIG. 21 shows the in vivo anti-tumor activity of h12F3 HGLF-mc-vc-MMAE or h12F3 HGLF-mp-dL AE-MMAE conjugates compared to their respective isotype ADC controls in the NCI-N87 gastric model.

Figure 22 shows a summary of tumor growth inhibition of h12F3-HGLF mc-vc-MMAE and mp-dLAE-MMAE in four xenograft tumor models including pancreas (HPAC), stomach (NCI-N87), ovary (CAOV3) and lung (SNU-2535). The average non-target ADC is shown as a dotted line.

Figure 23 shows the anti-tumor activity of ovarian patient-derived xenografts treated with h12F3-HDLF-mc-vc-MMAE. A) shows a summary of tumor growth inhibition of twelve xenografts, while B) and C) show examples of two conjugate-treated models compared to untreated cohorts.

FIG. 24 shows the binding affinity of h12F3 HGLF and HFLD to human ALPP and monkey ortholog ALPP.

FIG. 25 shows epitope mapping of h12F3 HGLF on HEK293 cells expressing chimeric rat/human ALPP.

Figure 26 shows sensorgrams showing the binding of h12F3, h12F3-HGLF-mc-vc-MMAE, h12F3-mp-dLAE-MMAE or positive control mAb (varies by row) to human Fc receptors (varies by column). The equilibrium dissociation constants are listed in the upper right corner of each sensorgram.

Figure 27 shows cell lysis of LoVo cells determined by chromium release assay after incubation of Na2[51Cr]O4 (Cr-51) labeled cells with NK cells in the presence of h12F3 HGLF, h12F3 HGLF-mc-vc-MMAE or h12F3 HGLF-mp-dLAE-MMAE conjugate.

Figure 28 shows the phagocytic activity of macrophages incubated with LoVo cells in the presence of h12F3 HGLF, h12F3 HGLF-mc-vc-MMAE or h12F3 HGLF-mp-dLAE-MMAE conjugates compared to positive (function blocking anti-CD47 antibody) or isotype controls.

FIG. 29 shows FcgRIII-mediated activation of luminescent reporters by h12F3 HGLF antibody and ADC.

FIG. 30 shows the activation of two signaling pathways involved in immunogenic cell death by two different concentrations of h12F3 HGLF-mc-vc-MMAE or h12F3 HGLF-mp-dLAE-MMAE conjugates.

31 shows ATP release in the culture medium of LoVo cells treated with 1 or 10 mg/ml h12F3 HGLF-mc-vc-MMAE or h12F3 HGLF-mp-dLAE-MMAE conjugates for 24 or 48 hours compared to free MMAE cytotoxin.

DETAILED DESCRIPTION

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

The techniques and procedures described or referenced herein are generally well understood and commonly used by those skilled in the art using routine methods, such as the widely used methods described in the following references: Sambrook et al., Molecular Cloning: A Laboratory Manual 4th Edition (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y.; Current Protocols In Molecular Biology (F. M. Ausubel et al., eds., (2003)); METHODS IN ENZYMOLOGY series (Academic Press, Inc.); PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames, and G. R. Taylor, eds. (1995)); Greenfield, ed. (2013) Antibodies, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Notebook (J.E.Cellis, 1998) Academic Press; Animal Cell Culture (R.I.Freshney, 1987); Introduction to Cell and Tissue Culture (J.P.Mather and P.E.Roberts, 1998) Plenum Press; Cell and Tissue Culture Laboratory Procedures (A.Doyle, J.B.Griffiths and D.G.Newell, 1993-8) J.Wiley and Sons; Handbook of Experimental Immunology (D.M.Weir and C.C.Blackwell, 1993); Gene Transfer Vectors for Mammalian Cells (J.M.Miller and M.P.Calos, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (J.E.Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty. ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean ed., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra ed., Harwood Academic Publishers, 1995); Cancer: Principles and Practice of Oncology (V.T. DeVita et al. ed., J.B. Lippincott Company, 1993); and updated versions thereof. Each of the foregoing references in this paragraph is incorporated herein by reference in its entirety.

I. Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd Edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 5th Edition, 2013, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, 2nd Edition, 2006, Oxford University Press, provide a general dictionary of many of the terms used in the present disclosure for those of skill in the art.

Unless otherwise required or clearly indicated by context, singular terms shall include pluralities and plural terms shall include the singular.

It should be understood that aspects and embodiments of the present invention described herein include "comprising," "consisting of," and/or "consisting essentially of" aspects and embodiments.

As used herein, unless otherwise indicated, the singular forms "a", "an", and "the" should be understood to mean "one or more" of any listed or enumerated components.

When used herein, the term "and/or" should be considered as a specific disclosure of each of two specific features or components with or without the other. Thus, the term "and/or" as used herein in phrases such as "A and/or B" is intended to include: "A and B," "A or B," "A" (alone) and "B" (alone). Similarly, the term "and/or" as used in phrases such as "A, B, and/or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

The term "about" refers to a value or composition within an acceptable error range for a particular value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. As will be appreciated by those skilled in the art, references herein to "about" values or parameters include (and describe) embodiments for the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

As described herein, any concentration range, percentage range, ratio range or integer range should be understood to include any integer within the range and fractional values thereof (such as tenths and hundredths of integers) where appropriate, unless otherwise specified.

When a trade name is used herein, reference to the trade name also refers to the product formulation, generic drug, and active pharmaceutical ingredient of the trade name product, unless the context indicates otherwise.

The terms "ALPP", "alkaline phosphatase", "placental alkaline phosphatase", "ALPase" or "PLAP" are used interchangeably herein and, unless otherwise indicated, include any naturally occurring variants (e.g., splice variants, allelic variants), isoforms, and vertebrate species homologs of human ALPP. The term encompasses "full-length", unprocessed ALPP as well as any form of ALPP produced by intracellular processing. The amino acid sequence of an exemplary human ALPP is provided in Uniprot ID: P05187 or RefSeq ID: NM_001632. The amino acid sequence of a specific example of a mature human ALPP protein is shown in SEQ ID NO: 2.

The terms "ALPPL2," "placental alkaline phosphatase 2," or "germ cell alkaline phosphatase" are used interchangeably herein and, unless otherwise indicated, include any naturally occurring variants (e.g., splice variants, allelic variants), isoforms, and vertebrate species homologs of human ALPPL2. The term encompasses "full-length," unprocessed ALPPL2 as well as any form of ALPPL2 produced by intracellular processing. The amino acid sequence of an exemplary human ALPPL2 is provided in Uniprot ID: P10696 or RefSeq ID: NM_031313. The amino acid sequence of a specific example of a mature human ALPPL2 protein is shown in SEQ ID NO: 4.

As used herein, an "antigen binding protein"("ABP") refers to any protein that binds to a specific target antigen, rather than a naturally occurring cognate ligand or a fragment of such a ligand that binds to a specific antigen. In the present application, the specific target antigen is ALPP and/or ALPPL2 or a fragment of ALPP and/or ALPPL2. "Antigen binding proteins" include proteins that comprise at least one antigen binding region or domain (e.g., at least one hypervariable region (HVR) or complementarity determining region (CDR) as defined herein). In some embodiments, the antigen binding protein comprises a scaffold, such as one or more polypeptides, in which one or more (e.g., 1, 2, 3, 4, 5 or 6) HVRs or CDRs as described herein are embedded and/or connected. In some antigen binding proteins, the HVRs or CDRs are embedded in a "framework" region that orients the HVRs or CDRs so that the appropriate antigen binding properties of the CDRs are achieved. For some antigen binding proteins, the scaffold is an immunoglobulin heavy chain and/or light chain from an antibody or fragment thereof. Other examples of scaffolds include, but are not limited to, human fibronectin (e.g., the 10th extracellular domain of human fibronectin III), neocarcinomatidylcholine CBM4-2, anti-catenin derived from lipocalin, designed ankyrin repeat domains (DARPins), protein-A domains (protein Z), Kunitz domains, Im9, TPR proteins, zinc finger domains, pVIII, GC4, transferrin, the B domain of SPA, Sac7d, the A-domain, the SH3 domain of Fyn kinase, and C-type lectin-like domains (see, e.g., Gebauer and Skerra (2009) Curr. Opin. Chem. Biol., 13:245-255; Binz et al. (2005) Nat. Biotech. 23:1257-1268; and Yu et al. (2017) Annu Rev Anal Chem 10:293-320, each of which is incorporated herein by reference in its entirety). Thus, antigen binding proteins include, but are not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies such as Synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions, and portions or fragments thereof. In some cases, the antigen binding protein is a functional fragment of an intact antibody (e.g., Fab, Fab', F(ab') ₂ , scFv, domain antibody, or minibody). Peptibodies are another example of antigen binding proteins. In some embodiments, the term "antigen binding protein" includes derivatives, such as antigen binding proteins that have been chemically modified, such as antigen binding proteins linked to another agent such as a label or a cytotoxic agent or a cytostatic agent (e.g., an antigen binding protein conjugate, such as an antibody drug conjugate).

As used herein, an "antigen-binding fragment" (or simply "fragment") or "antigen-binding domain" of an antigen-binding protein (e.g., an antibody) refers to one or more fragments of an antigen-binding protein (e.g., an antibody), regardless of how obtained or synthesized, that retain the ability to specifically bind to the antigen bound by the entire antigen-binding protein. Examples of antibody fragments include, but are not limited to, Fv; Fab; Fab';Fab'-SH;F(ab')₂;diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. An "Fv" fragment includes a non-covalently linked dimer of one heavy chain variable domain and one light chain variable domain. A "Fab" fragment includes, in addition to the heavy and light chain variable domains of the Fv fragment, the constant domain of the light chain and the first constant domain ( _CH1 ) of the heavy chain. A "F(ab') ₂ " fragment includes two Fab fragments linked by a disulfide bond near the hinge region.

The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues and include, but are not limited to, dimers, trimers, peptides, oligopeptides and polymers of amino acid residues. This definition encompasses full-length proteins and fragments thereof. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. The term "polypeptide" also refers to proteins including modifications to the native sequence, such as deletions, additions and substitutions (usually conservative in nature), as long as the protein retains the desired activity. The terms "polypeptide" and "protein" encompass ALPP and/or ALPPL2 antigen binding proteins, including antibodies, antibody fragments, or sequences having deletions, additions and/or substitutions of one or more amino acids of the antigen binding protein.

"Native sequence" or "naturally occurring" polypeptides include polypeptides having the same amino acid sequence as a polypeptide found in nature. Thus, a native sequence polypeptide can have the amino acid sequence of a naturally occurring polypeptide from any mammal. Such native sequence polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence" polypeptide specifically encompasses naturally occurring truncated or secreted forms of the polypeptide (e.g., an extracellular domain sequence), naturally occurring variant forms (e.g., alternatively spliced forms), and naturally occurring allelic variants of the polypeptide.

A polypeptide "variant" means a biologically active polypeptide (e.g., an antigen binding protein or antibody) having at least about 70%, 80%, or 90% amino acid sequence identity with a native or reference sequence polypeptide, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage sequence identity and not taking into account any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N- or C-terminus of the polypeptide. In some embodiments, the variant will have at least about 80% amino acid sequence identity. In some embodiments, the variant will have at least about 90% amino acid sequence identity. In some embodiments, the variant will have at least about 95% amino acid sequence identity with a native sequence polypeptide.

As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide, or antigen-binding protein (e.g., antibody) sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps (if necessary) to achieve maximum percentage sequence identity and not considering any conservative substitutions as part of sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved in various ways within the skill of the art, such as using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN ^™ (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequence. For example, the % sequence identity of a given amino acid sequence A to, with, or for a given amino acid sequence B (which can be alternatively expressed as a given amino acid sequence A having or comprising a certain % sequence identity to, with, or for a given amino acid sequence B) is calculated as follows:

Multiply 100 by the fraction X/Y

Where X is the number of amino acid residues scored as identical matches by the sequence in the alignment of A and B in this program, and where Y is the total number of amino acid residues in B. Unless otherwise specifically stated, all % amino acid sequence identity values used herein are calculated according to this formula using the ALIGN-2 computer program. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % sequence identity of A to B will not be equal to the % sequence identity of B to A.

The term "leader sequence" refers to a sequence of amino acid residues located at the N-terminus of a polypeptide that promotes secretion of the polypeptide from a mammalian cell. The leader sequence can be cleaved when the polypeptide is exported from a mammalian cell to form a mature protein. Leader sequences can be natural or synthetic, and they can be heterologous or homologous to the protein to which they are linked.

The term "immunoglobulin" refers to a class of structurally related glycoproteins that consist of two pairs of polypeptide chains, a pair of light (L) low molecular weight chains and a pair of heavy (H) chains, all four of which are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology (Paul, W. ed., 7th ed. Raven Press, NY (2013)). In brief, each heavy chain typically includes a heavy chain variable region (abbreviated herein as _VH or VH) and a heavy chain constant region ( _CH or CH). The heavy chain constant region typically includes three domains: _CH1 , _CH2 , and _CH3 . The heavy chains are typically interconnected by disulfide bonds in the so-called "hinge region". Each light chain typically includes a light chain variable region (abbreviated herein as _VL or VL) and a light chain constant region ( _CL or CL). The light chain constant region typically includes one domain, _CL . CL can be a κ (kappa) or λ (lambda) isotype. The terms "constant domain" and "constant region" are used interchangeably herein. Immunoglobulins can be derived from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses are also well known to those skilled in the art and include but are not limited to human IgG1, IgG2, IgG3 and IgG4. "Isotype" refers to the antibody class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region gene.

The term "antibody" is used in its broadest sense, and specifically encompasses, for example, monoclonal antibodies (including full-length or intact monoclonal antibodies), antibodies with multi-epitope or mono-epitope specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, as long as they exhibit the desired biological activity), single-chain antibodies, and fragments of the aforementioned antibodies, as described below. Antibodies can be human, humanized, chimeric and/or affinity matured, as well as antibodies from other species such as mice and rabbits. Thus, the term "antibody" includes, for example, polypeptide products of B cells within the immunoglobulin class of polypeptides, which are capable of binding to a specific molecular antigen and are composed of two pairs of identical polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), each amino terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxyl terminal portion of each chain includes a constant region. See, for example, Antibody Engineering (Borrebaeck, ed., 2nd ed., 1995); and Kuby, Immunology (3rd ed., 1997). The term "antibody" also includes, but is not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intracellular antibodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above antibodies, which refers to a portion of an antibody heavy chain and/or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab') ₂ fragments, Fv ₍ dsFv) linked by disulfide bonds, Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., antigen binding domains or molecules containing antigen binding sites (e.g., one or more CDRs of an antibody) that bind to an antigen. Such antibody fragments can be found in, e.g., Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics 22: 189-224; Plückthun and Skerra, 1989, Meth. Enzymol. 178: 497-515; and Day, Advanced Immunochemistry (2nd ed., 1990). The immunoglobulins disclosed herein can be immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

As used herein, the term "hypervariable region" or "HVR" refers to each region in an antibody variable domain where the sequence is highly variable. HVRs can form structurally defined loops ("hypervariable loops"). Typically, natural four-chain antibodies contain six HVRs; three in VH (H1, H2, H3), and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 show the greatest diversity among the six HVRs, and H3 in particular is considered to play a unique role in conferring good specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, Methods in Molecular Biology 248:1 -25 (Lo ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camel antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, eg, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

HVR generally comprises amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs), which have the highest sequence variability and/or participate in antigen recognition. A variety of schemes for defining the boundaries of a given CDR are known in the art. For example, the Kabat complementary determining region (CDR) is based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia refers to the position of the structural loop instead (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). AbM CDR represents a compromise between Kabat CDR and Chothia structural loops and is used by AbM antibody modeling software from Oxford Molecular. "Contact" CDR is based on an analysis of available complex crystal structures. Additional details regarding the foregoing scheme and other numbering conventions are provided in the following references: Al-Lazikani et al., (1997) J. Mol. Biol. 273:927-948 ("Chothia" numbering scheme); MacCallum et al., (1996) J. Mol. Biol. 262:732-745 (1996), ("Contact" numbering scheme); Lefranc M-P. et al., (2003) Dev. Comp. Immunol. 27:55-77 ("IMGT" numbering scheme); and Honegger A. and Pluckthun A. (2001) J. Mol/Biol. 309:657-70, (AHo numbering scheme).

In some embodiments, the HVR regions and associated sequences are identical to the CDR regions and associated sequences based on one of the aforementioned numbering conventions. Accordingly, the residues of exemplary HVRs and/or CDRs are summarized in Table 1 below.

Table 1: Summary of different CDR numbering schemes

ring IMGT Kabat AbM Chothia Contact CDR-H1 27-38 31-35 26-35 26-32 30-35 CDR-H2 56-65 50-65 50-58 52-56 47-58 CDR-H3 105-117 95-102 95-102 95-102 93-101 CDR-L1 27-38 24-34 24-34 24-34 30-36 CDR-L2 56-65 50-56 50-56 50-56 46-55 CDR-L3 105-117 89-97 89-97 89-97 89-96

In some embodiments, the HVR may comprise an extended HVR as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. For each of these definitions, the variable domain residues are numbered according to Kabat et al. (supra).

Unless otherwise indicated, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (such as a variable region) and the individual CDRs of an antibody or region thereof (e.g., CDR-H1, CDR-H2) are understood to encompass complementary determining regions defined by any known scheme as described above. In some cases, schemes for identifying one or more specific CDRs are specified, such as CDRs defined by the IMGT, Kabat, AbM, Chothia or Contact methods. In other cases, specific amino acid sequences of CDRs are given.

Thus, in some embodiments, the antigen binding protein comprises CDRs and/or HVRs as defined by the IMGT system. In other embodiments, the antigen binding protein comprises CDRs or HVRs as defined by the Kabat system. In other embodiments, the antigen binding protein comprises CDRs or HVRs as defined by the AbM system. In other embodiments, the antigen binding protein comprises CDRs or HVRs as defined by the Chothia system. In some embodiments, the antigen binding protein comprises HVR and/or CDR residues as identified in Figures 5 to 8 or as described elsewhere herein.

The term "variable region" or "variable domain" refers to the domain of an antigen-binding protein (e.g., antibody) heavy or light chain that participates in the binding of an antigen-binding protein (e.g., antibody) to an antigen. The variable region or domain of an antigen-binding protein such as an antibody's heavy and light chains (VH and VL, respectively) can be further subdivided into hypervariable regions (or hypervariable regions, which can be hypervariable in the form of loops defined by sequence and/or structure), such as hypervariable regions (HVRs) or complementary determining regions (CDRs), interspersed with more conservative regions, referred to as framework regions (FRs). Typically, there are three HVRs (HVR-H1, HVR-H2, HVR-H3) or CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region, and three HVRs (HVR-L1, HVR-L2, HVR-L3) or CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. "Framework region" and "FR" are known in the art and refer to the non-HVR or non-CDR portion of the heavy and light chain variable regions. Typically, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region, and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region. Within each VH and VL, three HVRs or CDRs and four FRs are typically arranged from amino terminus to carboxyl terminus in the following order: FR1, HVR1, FR2, HVR2, FR3, HVR3, FR4 in the case of HVRs, or FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 in the case of CDRs (see also Chothia and Lesk J. Mot. Biol., 195, 901-917 (1987)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using a VH or VL domain from an antibody that binds the antigen to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

As used herein, the term "heavy chain variable region" (VH) refers to a region comprising heavy chain HVR-H1, FR-H2, HVR-H2, FR-H3, and HVR-H3. For example, a heavy chain variable region may comprise heavy chain CDR-H1, FR-H2, CDR-H2, FR-H3, and CDR-H3. In some embodiments, the heavy chain variable region further comprises at least a portion of FR-H1 and/or at least a portion of FR-H4.

As used herein, the term "heavy chain constant region" refers to a region comprising at least three heavy chain constant domains, _CH1 , _CH2 , and _CH3 . Non-limiting exemplary heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody comprising a γ constant region is an IgG antibody, an antibody comprising a δ constant region is an IgD antibody, and an antibody comprising an α constant region is an IgA antibody. In addition, an antibody comprising a μ constant region is an IgM antibody, and an antibody comprising an ε constant region is an IgE antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (comprising a _γ1 constant region), IgG2 (comprising a _γ2 constant region), IgG3 (comprising a _γ3 constant region), and IgG4 (comprising a _γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (comprising an _α1 constant region) and IgA2 (comprising an _α2 constant region) antibodies; IgM antibodies include, but are not limited to, IgM1 and IgM2.

As used herein, the term "heavy chain" (HC) refers to a polypeptide comprising at least one heavy chain variable region, with or without a leader sequence. In some embodiments, the heavy chain comprises at least a portion of a heavy chain constant region. As used herein, the term "full-length heavy chain" refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

As used herein, the term "light chain variable region" (VL) refers to a region comprising light chain HVR-L1, FR-L2, HVR-L2, FR-L3, and HVR-L3. In some embodiments, the light chain variable region comprises light chain CDR-L1, FR-L2, CDR-L2, FR-L3, and CDR-L3. In some embodiments, the light chain variable region further comprises FR-L1 and/or FR-L4.

As used herein, the term "light chain constant region" refers to the region comprising the light chain constant domain, _CL . Non-limiting exemplary light chain constant regions include λ and κ.

As used herein, the term "light chain" (LC) refers to a polypeptide comprising at least one light chain variable region, with or without a leader sequence. In some embodiments, the light chain comprises at least a portion of a light chain constant region. As used herein, the term "full-length light chain" refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

When referring to residues in the constant region of an immunoglobulin heavy chain, the "EU numbering system" or "EU index" is generally used (e.g., the EU index reported in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991). The "EU index in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise indicated herein, references to residue numbers in an antibody constant domain refer to residue numbering by the EU numbering system.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies are highly specific for a single antigenic site. In contrast to polyclonal antibody preparations that can include different antibodies for different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

As used herein, a "bispecific" antibody refers to an antibody that has binding specificity for at least two different antigenic epitopes. In one embodiment, the epitopes are from the same antigen. In another embodiment, the epitopes are from two different antigens. Methods for preparing bispecific antibodies are known in the art. For example, bispecific antibodies can be produced by co-expression of two immunoglobulin heavy chain/light chain pairs. See, for example, Milstein et al., Nature 305:537-39 (1983). Alternatively, chemical linkage can be used to prepare bispecific antibodies. See, for example, Brennan et al., Science 229:81 (1985). Bispecific antibodies include bispecific antibody fragments. See, for example, Hollinger et al., Proc. Natl. Acad. Sci. U.S.A. 90:6444-48 (1993), Gruber et al., J. Immunol. 152:5368 (1994).

"Dual variable domain immunoglobulin" or "DVD-Ig" refers to a multivalent and multispecific binding protein as described, e.g., in DiGiammarino et al., Methods Mol. Biol. 899: 145-156, 2012, Jakob et al., MABs 5: 358-363, 2013, and U.S. Pat. Nos. 7,612,181, 8,258,268, 8,586,714, 8,716,450, 8,722,855, 8,735,546, and 8,822,645, each of which is incorporated by reference in its entirety.

"Dual affinity retargeting proteins" or "DARTs" are a form of bispecific antibodies in which the heavy variable domain of one antibody is linked to the light variable domain of another antibody and the two chains associate, and are described, for example, in Garber, Nature Reviews Drug Discovery 13:799-801, 2014.

"Bispecific T cell engager" or is a genetic fusion of two scFv fragments that produces a tandem scFv molecule and is described, for example, in Baeuerle et al., Cancer Res. 69:4941-4944, 2009.

As used herein, "chimeric antibody" refers to an antibody in which a portion of the heavy chain and/or light chain is derived from a specific source or species, while the rest of the heavy chain and/or light chain is derived from a different source or species. In some embodiments, a chimeric antibody refers to an antibody comprising at least one variable region from a first species (such as a mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (such as a human, cynomolgus monkey, etc.). In some embodiments, a chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, a chimeric antibody comprises at least one cynomolgus monkey variable region and at least one human constant region. In some embodiments, all variable regions of a chimeric antibody are from a first species, and all constant regions of a chimeric antibody are from a second species.

As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody comprising a human antibody constant domain and a non-human variable domain modified to comprise a high level of sequence homology with a human variable domain. This can be achieved by transplanting six non-human antibody complementary determining regions (CDRs) onto a homologous human receptor framework region (FR) (see WO92/22653 and EP0629240). In order to completely reconstruct the binding affinity and specificity of the parent antibody, it may be necessary to replace the framework residues from the parent antibody (i.e., non-human antibody) with human framework regions (reverse mutations). Structural homology modeling can help identify amino acid residues in the framework region that are important for the binding properties of antibodies. Therefore, a humanized antibody may comprise a non-human CDR sequence, mainly a human framework region, optionally comprising one or more amino acid reverse mutations to a non-human amino acid sequence; and a fully human constant region. Optionally, additional amino acid modifications may be applied, not necessarily reverse mutations, to obtain humanized antibodies with preferred features such as affinity and biochemical properties.

As used herein, "human antibody" refers to antibodies produced in humans, antibodies produced in non-human animals containing human immunoglobulin genes, such as And antibodies selected using in vitro methods such as phage display, wherein the antibody library is based on human immunoglobulin sequences. "Human antibody" is an antibody with a variable region, wherein FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. Human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been transplanted to human framework sequences. The terms "human antibody" and "fully human antibody" are used synonymously.

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

An "affinity matured" antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody that does not have such alterations, such alterations resulting in an improvement in the affinity of the antibody for the antigen. In some examples, an affinity matured antibody refers to an antibody with one or more alterations in one or more complementarity determining regions (CDRs) compared to a parent antibody that does not have such alterations, such alterations resulting in an improvement in the affinity of the antibody for the antigen.

The term "derivative" refers to a molecule (e.g., antigen-binding protein, such as an antibody or its fragment) that includes a chemical modification other than the insertion, deletion or substitution of an amino acid (or nucleic acid). In certain embodiments, the derivative comprises a covalent modification, including but not limited to chemical bonding with a polymer, a lipid or other organic or inorganic part. In certain embodiments, the derivative of a specific antigen-binding protein may have a longer circulation half-life than an antigen-binding protein that is not chemically modified. In certain embodiments, the derivative may have improved targeting capabilities for desired cells, tissues and/or organs. In some embodiments, the derivative of an antigen-binding protein is covalently modified to include one or more polymers, including but not limited to monomethoxy-polyethylene glycol, dextran, cellulose or other carbohydrate-based polymers, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylene polyols (e.g., glycerol) and polyvinyl alcohol, and mixtures of such polymers. See, e.g., U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337.

As used herein, the term "epitope" refers to a site on an antigen (e.g., ALPP or ALPPL2) to which an antigen-binding protein (e.g., an antibody or fragment thereof) that targets the antigen binds. An epitope is typically composed of chemically active surface groups of molecules such as amino acids, polypeptides, sugar side chains, phosphoryl or sulfonyl groups, and has specific three-dimensional structural characteristics and specific charge characteristics. An epitope can be formed by continuous or discontinuous amino acids of an antigen juxtaposed by tertiary folding. Epitopes formed by continuous residues are typically retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are typically lost when treated with denaturing solvents. In certain embodiments, an epitope may include, but is not limited to, at least 3, at least 4, at least 5, at least 6, or at least 7 amino acids in a unique spatial arrangement. In some embodiments, an epitope refers to 3-5, 4-6, or 8-10 amino acids in a unique spatial conformation. In other embodiments, the length of the epitope is less than 20 amino acids, less than 15 amino acids, or less than 12 amino acids, less than 10 amino acids, or less than 8 amino acids. In one embodiment, the epitope of the anti-ALPP/ALPPL2 antibody of the present invention comprises SEQ ID NO: 73 and/or SEQ ID NO: 74. The epitope may comprise amino acid residues that are directly involved in binding (also referred to as the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding, including amino acid residues that are effectively blocked or covered by the antigen binding molecule (i.e., the amino acid is within the footprint of the antigen binding molecule). Methods for determining the spatial conformation of epitopes include, for example, x-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, edited by G.E. Morris (1996)). Once the desired epitope of an antigen is determined, an antigen binding protein (e.g., an antibody or fragment thereof) directed against the epitope can be generated using established techniques. The resulting antigen binding proteins can then be screened in a competition assay to identify antigen binding proteins that bind to the same or overlapping epitopes. Methods for classifying antibodies based on cross-competition studies are described in WO 03/48731.

A "non-linear epitope" or "conformational epitope" comprises a discontinuous polypeptide, amino acid and/or carbohydrate within an antigenic protein to which an antibody specific for the epitope binds.

A "linear epitope" comprises contiguous polypeptides, amino acids and/or carbohydrates within an antigenic protein to which an antigen binding protein (eg, an antibody or fragment thereof) specific for the epitope binds.

When used in the context of antigen binding proteins (e.g., antibodies or fragments thereof) that compete for the same epitope, the term "competition" means competition between antigen binding proteins, as determined by an assay in which the antigen binding protein (e.g., antibody or fragment thereof) being tested (e.g., test antibody) blocks or inhibits (partially or completely) specific binding of a reference antigen binding protein (e.g., reference antibody) to a common antigen (e.g., ALPP or ALPPL2 or fragment thereof). Various types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, including various label-free biosensor methods, such as surface plasmon resonance (SPR) analysis (see, e.g., Abdiche et al., 2009, Anal. Biochem. 386: 172-180; Abdiche et al., 2012, J. Immunol Methods 382: 101-116; and Abdiche et al., 2014 PLoS One 9: e92451). Other assays that can be used include: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619); solid phase direct labeling assay, solid phase direct labeling sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labeling RIA using 1-125 labeling (see, e.g., Morel et al., 1988, Mol. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virology 176:546-552); direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, the test antigen binding protein is present in excess (e.g., at least 2x, 5x, 10x, 20x or 100x). Typically, when the competitive antigen binding protein is present in excess, it will inhibit the specific binding of the reference antigen binding protein to the common antigen by at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%. In the case where each antigen binding protein (e.g., an antibody or fragment thereof) detectably inhibits the binding of another antigen binding protein to its cognate epitope, whether to the same, greater or lesser extent, the antigen binding proteins are referred to as "cross-competing" with each other to bind to their corresponding epitopes or "cross-blocking" each other. Typically, such cross-competition studies are performed using the conditions and methods described above for competition studies, and the degree of blocking in each manner is at least 30%, at least 40% or at least 50%. Additional methods and details of the methods for identifying competing antigen binding proteins are described in the Examples herein.

"Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule X for its partner Y can often be expressed in terms of a dissociation constant ( _Kd ). Affinity can be measured by common methods known in the art, including those described herein.

An "affinity matured" antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody that does not have such alterations, such alterations resulting in an improvement in the affinity of the antibody for the antigen. In some examples, an affinity matured antibody refers to an antibody with one or more alterations in one or more complementarity determining regions (CDRs) compared to a parent antibody that does not have such alterations, such alterations resulting in an improvement in the affinity of the antibody for the antigen.

As used herein, the term "specifically binds,""binds," or simply "binds," or other related terms in the context of an antigen binding protein binding to its target antigen means that the antigen binding protein substantially exhibits background binding to non-target molecules. However, an antigen binding protein that specifically binds to a target antigen (e.g., ALPP and/or ALPPL2) may cross-react with ALPP and/or ALPPL2 proteins from different species. Typically, an ALPP/ALPPL2 antigen binding protein specifically binds to ^human ALPP and/or ALPPL2 when the dissociation constant ( _KD ) is ^10-7 M or less, such as about ^10-8 M or less, such as about ^10-9 M or less, about ^10-10 M or less, about 10-11 M or less, or about ^10-12 or even less, as measured by surface plasmon resonance (SPR) technology (e.g., BIACore, GE-Healthcare Uppsala, Sweden) using an antibody as a ligand and an antigen as an analyte.

As used herein, the term " _KD " (M) refers to the dissociation equilibrium constant of a particular antigen binding protein-antigen interaction (e.g., an antibody-antigen interaction). As used herein, affinity is inversely related to _KD , such that a higher affinity is intended to refer to a smaller _KD , and a lower affinity is intended to refer to a larger _KD .

"Antibody-drug-conjugate" or "ADC" for short refers to an antibody conjugated to a cytotoxic or cytostatic agent. The antibody-drug-conjugate typically binds to a target antigen (e.g., ALPP and/or ALPPL2) on the cell surface, and then the antibody-drug-conjugate is internalized into the cell, where the drug is released.

The abbreviations "vc" and "val-cit" refer to the dipeptide valine-citrulline.

The abbreviation LAE refers to the tripeptide linker Leucine-Alanine-Glutamic acid. The abbreviation dLAE refers to the tripeptide linker D-Leucine-Alanine-Glutamic acid, wherein the Leucine in the tripeptide linker is in the D-configuration.

The abbreviation VKG refers to the tripeptide linker valine-lysine-glycine.

The abbreviation "PABC" refers to a self-consumable spacer:

The abbreviation "mc" refers to extended maleimidocaproyl:

The abbreviation "mp" refers to extended maleimidopropionyl:

As used herein, a "PEG unit" is an organic moiety comprising repeating ethylene-oxy subunits (PEG or PEG subunits), and can be polydisperse, monodisperse or discrete (i.e., having a discrete number of ethylene-oxy subunits). Polydisperse PEGs are heterogeneous mixtures of size and molecular weight, while monodisperse PEGs are typically purified from heterogeneous mixtures, thus providing a single chain length and molecular weight. Preferred PEG units include discrete PEGs, compounds synthesized in a stepwise manner rather than by a polymerization process. Discrete PEGs provide individual molecules with defined and specified chain lengths.

The PEG units provided herein include one or more polyethylene glycol chains, each of which includes one or more ethyleneoxy subunits covalently linked to each other. The polyethylene glycol chains can be linked together, for example, in a straight chain, a branched chain, or a star-shaped configuration. Typically, before incorporation into the camptothecin conjugate, at least one polyethylene glycol chain is derivatized at one end with an alkyl moiety substituted with an electrophilic group to covalently link to the carbamate nitrogen (i.e., an example representing R) of the methylene carbamate unit. Typically, the terminal ethyleneoxy subunit in each polyethylene glycol chain that does not participate in the covalent link to the rest of the linker unit is modified with a PEG end-capping unit (typically an optionally substituted alkyl, such as -CH ₃ , CH ₂ CH ₃ or CH ₂ CH ₂ CO ₂ H). A preferred PEG unit has a single polyethylene glycol chain having 2 to 24 -CH ₂ CH ₂ O- subunits covalently linked in series and terminated at one end with a PEG end-capping unit.

"Cytotoxic effect" refers to the depletion, elimination and/or killing of target cells.

"Cytotoxic agent" refers to an agent that has a cytotoxic effect on cells.

"Cytostatic effect" refers to the inhibition of cell proliferation.

"Cytostatic agents" refer to agents that have a cytostatic effect on cells, thereby inhibiting the growth and/or expansion of a specific cell subpopulation. The cytostatic agent may be conjugated to an antibody or administered in combination with an antibody.

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

A "functional Fc region" has the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include Fc receptor binding; C1q binding; complement dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require an Fc region in combination with a binding domain (e.g., an antibody variable domain), and can be assessed using a variety of assays.

A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of a naturally occurring Fc region. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes), native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, and naturally occurring variants thereof.

A "variant Fc region" comprises an amino acid sequence which differs from a native sequence Fc region by virtue of at least one amino acid modification.

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, FcγR is a natural human FcR. In some embodiments, FcR is an FcR that binds to IgG antibodies (gamma receptors) and includes receptors of FcγRI, FcγRII, and FcγRIII subclasses (including allelic variants and alternative splicing forms of these receptors). FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences, the main difference being their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcRs are reviewed in, for example, Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term "FcR" herein encompasses other FcRs, including those to be identified in the future. The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulating the homeostasis of immunoglobulins. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.).

"Effector function" refers to the biological activity attributable to the Fc region of an antibody, which varies with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. Such functions can be affected by, for example, binding of the Fc effector domain to Fc receptors on immune cells with phagocytic or lytic activity or by binding of the Fc effector domain to components of the complement system. Typically, the effects mediated by Fc binding to cells or complement components lead to inhibition and/or depletion of CD33-targeted cells. The Fc region of an antibody can recruit cells expressing Fc receptors (FcRs) and juxtapose them with antibody-coated target cells. Cells expressing surface FcRs of IgG including FcγRIII (CD16), FcγRII (CD32), and FcγRIII (CD64) can serve as effector cells that destroy IgG-coated cells. Such effector cells include monocytes, macrophages, natural killer (NK) cells, neutrophils and eosinophils. IgG binding to FcγR activates antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP). ADCC is mediated by CD16 ⁺ effector cells through secretion of membrane pore-forming proteins and proteases, while phagocytosis is mediated by CD32 ⁺ and CD64 ⁺ effector cells (see, e.g., Fundamental Immunology, 4th edition, Paul edited, Lippincott-Raven, NY, 1997, chapters 3, 17 and 30; Uchida et al., 2004, J. Exp. Med. 199: 1659-69; Akewanlop et al., 2001, Cancer Res. 61: 4061-65; Watanabe et al., 1999, Breast Cancer Res. Treat. 53: 199-207.

"Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. In certain embodiments, the cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources, such as from blood.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cytotoxic mechanism in which the Fc region of an antibody bound to an antigen on the cell surface of a target cell interacts with Fc receptors (FcRs) present on certain cytotoxic effector cells (e.g., NK cells, neutrophils, and macrophages). This interaction enables these cytotoxic effector cells to subsequently kill target cells with cytotoxins. Primary cells NK cells that mediate ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). In order to evaluate the ADCC activity of a target molecule, an in vitro ADCC assay can be performed, such as the assay described in U.S. Pat. No. 5,500,362 or 5,821,337 or U.S. Pat. No. 6,737,056 (Presta). Useful effector cells for such determinations include PBMC and NK cells. The ADCC activity of the target molecule can also be evaluated in vivo, for example in an animal model, such as the animal model disclosed in Clynes et al. Proc. Natl. Acad. Sci. (USA) 95: 652-656 (1998). Other polypeptide variants with altered Fc region amino acid sequences (polypeptides with variant Fc regions) and increased or decreased ADCC activity are described in, for example, U.S. Patent No. 7,923,538 and U.S. Patent No. 7,994,290.

"Complement dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by binding of the first component of the complement system (C1q) to the Fc region of antibodies (of the appropriate subclass) that bind to their cognate antigens on the target cell. This binding activates a series of enzymatic reactions that ultimately form pores in the target cell membrane and subsequently lead to cell death. Activation of complement may also result in the deposition of complement components on the target cell surface, which promote ADCC by binding to complement receptors (e.g., CR3) on leukocytes. To assess complement activation, a CDC assay may be performed, such as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). Polypeptide variants with altered Fc region amino acid sequences (polypeptides such as antibodies with variant Fc regions) and increased or decreased CIq binding ability are described in, e.g., U.S. Pat. No. 6,194,551 Bl, U.S. Pat. No. 7,923,538, U.S. Pat. No. 7,994,290, and WO 1999/51642. See also, e.g., Idusogie et al., J. Immunol. 164:4178-4184 (2000).

The term "antibody-dependent cellular phagocytosis," or "ADCP" for short, refers to the process by which antibody-coated cells are internalized in whole or in part by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind the Fc region of the Ig.

A polypeptide variant (e.g., antibody) with "altered" FcR binding affinity or ADCC activity is a polypeptide variant that has enhanced or diminished FcR binding activity and/or ADCC activity compared to a parent polypeptide or to a polypeptide comprising a native sequence Fc region. A polypeptide variant that "exhibits increased binding to an FcR" binds to at least one FcR with better affinity than the parent polypeptide. A polypeptide variant that "exhibits decreased binding to an FcR" binds to at least one FcR with lower affinity than the parent polypeptide. In some embodiments, such variants that exhibit decreased binding to an FcR may have little or no significant binding to an FcR, e.g., 0-20% binding to an FcR, compared to a native sequence IgG Fc region.

The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to polymers of nucleotides of any length. Such nucleotide polymers may contain natural and/or non-natural nucleotides and include, but are not limited to, DNA, RNA and PNA. "Nucleic acid sequence" refers to a linear sequence of nucleotides comprising a nucleic acid molecule or polynucleotide.

The term "vector" means any molecule or entity (e.g., a nucleic acid, plasmid, phage, or virus) used to transfer a nucleic acid molecule into a host cell. A vector generally includes a nucleic acid molecule that is engineered to contain one or more cloned polynucleotides encoding one or more target polypeptides that can be propagated in a host cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, and expression vectors, such as recombinant expression vectors. A vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., promoters and/or enhancers) that regulate expression of a target polypeptide, and/or one or more selectable marker genes. The term includes vectors that are self-replicating nucleic acid molecules as well as vectors that are incorporated into the genome of a host cell into which they have been introduced.

The term "expression vector" refers to a vector suitable for transforming a host cell and can be used to express a polypeptide of interest in the host cell.

The terms "host cell" or "host cell line" are used interchangeably herein and refer to a cell or cell population that can be or has been a recipient of a vector or isolated polynucleotide. Host cells can be prokaryotic or eukaryotic. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, Cells (Crucell) and 293 and CHO cells, and their derivatives, such as 293-6E and DG44 cells, respectively. These terms refer not only to the original cell, but also to the descendants of such cells. Due to, for example, mutations or environmental influences, certain modifications may occur in the descendants. These descendants are also included in the term, as long as the cell has the same function or biological activity as the original cell.

The term "control sequence" refers to a polynucleotide sequence that can affect the expression and processing of the coding sequence connected thereto. The nature of such control sequences can depend on the host organism. In a specific embodiment, the control sequence of a prokaryote can include a promoter, a ribosome binding site, and a transcription termination sequence. The control sequence of a eukaryote can include, for example, a promoter, a transcription enhancer sequence, and a transcription termination sequence that comprise one or more transcription factor recognition sites." control sequence" can include a leader sequence and/or a fusion partner sequence.

As used herein, "operably linked" means that the components to which the term is applied are in a relationship that allows them to perform their inherent functions under appropriate conditions. For example, a control sequence in a vector that is "operably linked" to a protein coding sequence is linked to it so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence. In the case where two coding sequences are operably linked, the phrase means that the two DNA fragments or coding sequences are linked so that the amino acid sequences encoded by the two fragments remain in frame.

The term "transfection" refers to the uptake of foreign or exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA is introduced into the cell membrane. Many transfection techniques are well known in the art and disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into a suitable host cell.

The term "transformation" refers to a change in the genetic characteristics of a cell, and a cell is transformed when it is modified to contain new DNA or RNA. For example, a cell is transformed by introducing new genetic material through transfection, transduction or other techniques so that it is genetically modified from its native state. After transfection or transduction, the transforming DNA can be recombined with the cell's DNA by physically integrating into the cell's chromosome, or can be temporarily maintained as a free element without being replicated, or can be replicated independently as a plasmid. When the transforming DNA replicates as the cell divides, the cell is considered to have been "stably transformed."

As used herein, the term "isolated" refers to a molecule that has been separated from at least some of the components that are typically found or produced in nature. For example, when a polypeptide is separated from at least some of the components of the cell that produces it, it is referred to as "isolated". When a polypeptide is secreted by a cell after expression, the supernatant containing the polypeptide is physically separated from the cell that produces the polypeptide and is considered to be "isolated" the polypeptide. Similarly, when a polynucleotide is not part of a larger polynucleotide that is typically found in nature (e.g., genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide), or is separated from at least some of the components of the cell that produces the polynucleotide (e.g., in the case of an RNA polynucleotide), the polynucleotide is referred to as "isolated". Therefore, a DNA polynucleotide contained in a vector within a host cell can be referred to as "isolated".

The terms "individual", "subject" or patient are used interchangeably herein and refer to animals, such as mammals. In some embodiments, methods for treating mammals are provided, including but not limited to humans, rodents, apes, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sports animals, and mammalian pets. In some cases, an "individual" or "subject" is a human. In some instances, an "individual" or "subject" refers to an individual or subject (e.g., a human) in need of treatment for a disease or condition.

As used herein, "disease" or "disorder" refers to a condition in which treatment is required.

As used herein, "cancer" and "tumor" are interchangeable terms that refer to any abnormal cell or tissue growth or proliferation in an animal. As used herein, the terms "cancer" and "tumor" encompass solid cancers and blood/lymphatic cancers, and also encompass malignant, pre-malignant, and benign growths, such as dysplasia. Solid tumors are abnormal growths or masses of tissue that do not usually contain cysts or fluid areas. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific, non-limiting examples of such cancers include squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, brain cancer, endometrial cancer, testicular cancer, bile duct cancer, gallbladder cancer, gastric cancer, melanoma, and various types of head and neck cancer.

"Tumor burden", also known as "tumor load", refers to the total amount of tumor material distributed throughout the body. Tumor burden refers to the total number of cancer cells or the total size of the tumor throughout the body (including lymph nodes and bone stenosis). Tumor burden can be determined by a variety of methods known in the art, such as by measuring the size of the tumor when removed from the subject, such as using calipers, or using imaging techniques such as ultrasound, bone scans, computed tomography (CT) or magnetic resonance imaging (MRI) scans when in vivo.

The terms "metastatic cancer" and "metastatic disease" mean cancer that has spread from its site of origin to another part of the body, such as to regional lymph nodes or to distant sites.

The terms "advanced cancer," "locally advanced cancer," "advanced disease," and "locally advanced disease" refer to cancer that has extended through the capsule of the relevant tissue. Surgery is generally not recommended for patients with locally advanced disease, and these patients have significantly poorer outcomes than patients with clinically localized (organ-confined) cancer.

As used herein, "treatment" is a method for obtaining a beneficial or desired clinical outcome. As used herein, "treatment" encompasses any administration or application of a therapeutic agent for a disease in a mammal (including a human). Beneficial or desired clinical outcomes include, but are not limited to, any one or more of the following: alleviating one or more symptoms, alleviating the extent of the disease, preventing or delaying the spread of the disease (e.g., metastasis, such as metastasis to the lungs or lymph nodes), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, inhibiting the progression of the disease or the disease, inhibiting or slowing the disease or its progression, preventing its development and alleviating (whether in part or in whole). "Treatment" also includes reducing the pathological consequences of proliferative diseases.

In the context of cancer, the term "treat" includes any or all of the following: inhibiting the growth of cancer cells, inhibiting the replication of cancer cells, reducing the number of cancer cells, reducing the rate of cancer cell infiltration into surrounding organs, reducing the rate or extent of tumor metastasis, reducing the overall tumor burden, and ameliorating one or more symptoms associated with cancer.

In the context of autoimmune disease, the term "treating" includes any or all of the following: preventing the replication of cells associated with the autoimmune disease state (including but not limited to cells capable of producing autoimmune antibodies), reducing the autoimmune antibody burden, and ameliorating one or more symptoms of the autoimmune disease.

In the context of an infectious disease, the term "treating" includes any or all of preventing the growth, proliferation or replication of the pathogen causing the infectious disease and ameliorating one or more symptoms of the infectious disease.

The term "inhibit" refers to a reduction or cessation of any phenotypic characteristic, or to a reduction or cessation of the incidence, degree or likelihood of the characteristic. "Reduce" or "inhibit" refers to a reduction, decrease or prevention of activity, function and/or amount compared to a reference. In certain embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 20% or greater. In another embodiment, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 50% or greater. In yet another embodiment, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95% or greater.

As used herein, "reference" refers to any sample, standard or level used for comparison purposes. References can be obtained from healthy and/or non-diseased samples. In some instances, references can be obtained from untreated samples. In some instances, references are obtained from non-diseased or untreated samples of individual subjects. In some instances, references are obtained from one or more healthy individuals who are not subjects or patients.

As used herein, "delaying the development of a disease" means delaying, hindering, slowing, slowing, stabilizing, inhibiting and/or postponing the development of a disease (such as cancer). Such a delay may be of varying lengths of time, depending on the history of the disease and/or the individual being treated. As will be apparent to one skilled in the art, a sufficient or significant delay may actually encompass prevention, in that the individual does not develop the disease. For example, the development of an advanced cancer, such as metastasis, may be delayed.

As used herein, "prevention" includes providing prevention with respect to the occurrence or recurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease.

As used herein, "inhibiting" a function or activity means reducing the function or activity when compared to the same conditions other than the conditions or parameters of interest, or alternatively, compared to another condition. For example, an antibody that inhibits tumor growth reduces the growth rate of a tumor compared to the growth rate of the tumor in the absence of the antibody.

An "effective amount" or "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of a drug or agent that provides a therapeutic effect when used alone or in combination with another therapeutic agent, such as protecting a subject from the onset of a disease or promoting regression of a disease, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of impairment or disability due to disease affliction. The ability of a therapeutic agent to promote regression of a disease can be assessed using a variety of methods known to the skilled artisan, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by measuring the activity of the agent in an in vitro assay.

For example, for the treatment of tumors, in some embodiments, a therapeutically effective amount of an anticancer agent inhibits cell growth or tumor growth in a treated subject (e.g., one or more treated subjects) by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% relative to an untreated subject (e.g., one or more untreated subjects). In some embodiments, a therapeutically effective amount of an anticancer agent inhibits cell growth or tumor growth in a treated subject (e.g., one or more treated subjects) by 100% relative to an untreated subject (e.g., one or more untreated subjects). In other embodiments of the present disclosure, tumor regression can be observed and lasts for a period of at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, or at least about 60 days.

The therapeutically effective amount of a drug includes a "prophylactically effective amount," which is any amount of a drug that inhibits the development or recurrence of cancer when administered alone or in combination with an anticancer agent to a subject at risk of developing cancer (e.g., a subject with a precancerous condition) or a subject suffering from a recurrence of cancer. In some embodiments, a prophylactically effective amount completely prevents the development or recurrence of cancer. "Inhibiting" the development or recurrence of cancer means reducing the likelihood of cancer development or recurrence, or completely preventing the development or recurrence of cancer.

As used herein, "subtherapeutic dose" means a dose of a therapeutic compound that is lower than the usual or typical dose of the therapeutic compound when administered alone for the treatment of a hyperproliferative disease (eg, cancer).

"Administering" refers to physically introducing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion (e.g., intravenous infusion). Administration can also be performed, for example, once, multiple times and/or over one or more extended time periods.

As used herein, the term "monotherapy" means that the anti-ALPP/ALPPL2 antibody or ADC of the invention is the only anti-cancer agent administered to a subject during a treatment cycle. However, other therapeutic agents may be administered to the subject. For example, anti-inflammatory agents or other agents administered to a subject with cancer to treat symptoms associated with the cancer, but not the underlying cancer itself, including, for example, inflammation, pain, weight loss, and general discomfort, may be administered during a monotherapy period.

Administration "in combination with one or more additional therapeutic agents" includes simultaneous (concurrent) and serial or sequential administration in any order.

The term "simultaneous" is used herein to refer to the administration of two or more therapeutic agents, wherein at least a portion of the administration overlaps in time or wherein the administration of one therapeutic agent falls within a shorter time period relative to the administration of another therapeutic agent. For example, two or more therapeutic agents are administered simultaneously or at a time interval of no more than about 60 minutes, such as no more than about any of 30, 15, 10, 5, or 1 minute.

The term "sequentially" is used herein to refer to the administration of two or more therapeutic agents, wherein the administration of one or more agents continues after the administration of one or more other agents has ceased. For example, the administration of two or more therapeutic agents is administered at intervals exceeding about 15 minutes, such as any of about 20, 30, 40, 50 or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks or 1 month or longer.

The term "chemotherapeutic agent" refers to all compounds that are effective in inhibiting tumor growth. Non-limiting examples of chemotherapeutic agents include alkylating agents (e.g., nitrogen mustards, ethyleneimine compounds, and alkyl sulfonates); antimetabolites (e.g., folic acid, purine or pyrimidine antagonists); mitotic inhibitors (e.g., anti-tubulin agents such as vinca alkaloids, auristatins, and derivatives of podophyllotoxin); cytotoxic antibiotics; compounds that damage or interfere with DNA expression or replication (e.g., DNA minor groove binders); and growth factor receptor antagonists and cytotoxic agents or cell growth inhibitors.

The phrase "pharmaceutically acceptable" means that a substance or composition is chemically and/or toxicologically compatible with the other ingredients making up the formulation and/or the subject to be treated therewith.

The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to preparations that are in a form that permits the biological activity of the active ingredient to be effective and that do not contain additional components that are unacceptably toxic to a subject to which the formulation would be administered. Such preparations may be sterile.

"Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation adjuvant or carrier conventionally used with a therapeutic agent in the art, which together constitute a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration employed and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation employed.

As used herein, the phrase "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound of the invention. Exemplary salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisates, fumarates, gluconates, glucuronates, saccharates, formates, benzoates, glutamates, methanesulfonates "mesylate", ethanesulfonates, benzenesulfonates, p-toluenesulfonates, pamoates (i.e., 4,4'-methylene-bis-(2-hydroxy-3-naphthoate)), alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Cases where multiple charged atoms are part of a pharmaceutically acceptable salt may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions.

Various aspects of the disclosure are described in more detail in the following sections.

II. Overview

The present invention provides an antibody-drug conjugate, including an anti-ALPP antibody conjugated to vcMMAE (sometimes referred to herein as mc-vc-PABC-MMAE or mc-vc-MMAE) or dLAE-MMAE (sometimes referred to herein as mp-dLAE-PABC-MMAE or mp-dLAE-MMAE), which is particularly effective in killing cells expressing ALPP+. The present invention also provides an antibody-drug conjugate, including an anti-ALPPL2 antibody conjugated to vcMMAE or dLAE-MMAE, which is particularly effective in killing cells expressing ALPPL2+. In a preferred embodiment, the present invention provides an antibody (anti-ALPP/ALPPL2 antibody) that binds both ALPP and ALPPL2 conjugated to vcMMAE or dLAE-MMAE, which is particularly effective in killing cells expressing both ALPP+ and ALPPL2+. Both ALPP and ALPPL2 have been shown to be expressed in a variety of cancers, including ovarian cancer, lung cancer, endometrial cancer, testicular cancer, and gastric cancer.

Provided herein are antigen binding proteins (ABPs) that bind to ALPP/ALPPL2, including antigen binding fragments thereof (e.g., antibodies and antigen binding fragments thereof). In some embodiments, the antigen binding proteins and fragments contain an antigen binding domain that specifically binds to ALPP (including human ALPP) (e.g., SEQ ID NO: 2). In some embodiments, the antigen binding proteins and fragments contain an antigen binding domain that specifically binds to ALPPL2 (including human ALPPL2) (e.g., SEQ ID NO: 4). In some embodiments, the antigen binding proteins and fragments contain an antigen binding domain that specifically binds to both ALPP and ALPPL2 (including human ALPP (e.g., SEQ ID NO: 2) and human ALPPL2 (e.g., SEQ ID NO: 4)).

III. Anti-ALPP/ALPPL2 Antigen Binding Proteins, Including Fragments

A variety of antigen-binding proteins are provided herein and described in more detail below. Antigen-binding proteins disclosed herein generally include a scaffold, such as one or more polypeptides, in which one or more (e.g., 1, 2, 3, 4, 5 or 6) hypervariable regions (HVRs) or complementary determining regions (CDRs) are embedded, transplanted and/or connected. In some antigen-binding proteins, HVRs or CDRs are embedded, transplanted or connected to a "framework" region that directs HVRs or CDRs so as to achieve the appropriate antigen-binding properties of HVRs or CDRs. In some embodiments, the antigen-binding proteins include one or more VH and/or VL domains.

In some antigen binding proteins, HVR or CDR sequences are embedded, transplanted or connected to a protein scaffold or other biocompatible polymer. In some embodiments, the antigen binding protein is an antibody or is derived from an antibody. Therefore, the antigen binding proteins provided include but are not limited to monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions, antibody conjugates, and parts or fragments of each of the foregoing. Examples of antigen binding proteins provided herein as fragments include but are not limited to Fab, Fab', F(ab') ₂ , scFv and domain antibodies.

In some embodiments, the antigen binding protein binds to ALPP with an affinity (e.g., _KD ) of less than 10 nM, 5 nM, 2 nM, 1 nM, 500 pM, 250 pM, 200 pM, 150 pM, 130 pM, 100 pM, 50 pM, 25 pM, 10 pM, or 1 pM. In some embodiments, the ABP binds to ALPP with an affinity between 1 pM-10 nM, 1 pM-5 nM, 1 pM-1 nM, 100-200 pM, 100-150 pM, or 120-140 pM. In some embodiments, the binding affinity to ALPP is determined according to the assays described in the Examples. In some embodiments, the antigen binding protein binds to ALPPL2 with an affinity (e.g., _KD ) of less than 10 nM, 5 nM, 2 nM, 1 nM, 500 pM, 250 pM, 100 pM, 50 pM, 44 pM, 25 pM, 10 pM, or 1 pM. In some embodiments, the ABP binds to ALPPL2 with an affinity between 0.1 pM-5 nM, 0.1 pM-1 nM, 1 pM-1 nM, 1-100 pM, 1-75 pM, 10-75 pM, 10-50 pM, or 30-50 pM. In some embodiments, the binding affinity for ALPPL2 is determined according to the assays described in the Examples.

A. Exemplary Antigen Binding Proteins, Including Fragments

In one embodiment, the antigen binding proteins of the invention include the antibody 12F3 described in the Examples herein. In some embodiments, the antigen binding proteins of the invention include murine, chimeric, humanized and/or human 12F3 antibodies.

In one embodiment, the antigen binding protein disclosed herein comprises CDR-H1, CDR-H2, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 56-58 or 60-62, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 63-65 or 68-70, respectively. In another embodiment, the antigen binding protein disclosed herein comprises CDR-H1, CDR-H2, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 56-58, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 63-65, respectively, wherein the CDRs are determined by Kabat. In another embodiment, the antigen binding protein disclosed herein comprises CDR-H1, CDR-H2, CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 60-62, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 68-70, respectively, wherein the CDRs are determined by IMGT.

In another embodiment, the antigen binding protein disclosed herein comprises: a VH comprising the amino acid sequence of SEQ ID NO:15 and a VL comprising the amino acid sequence of SEQ ID NO:30.

In another embodiment, the antigen binding protein disclosed herein comprises: a HC comprising the amino acid sequence of SEQ ID NO:40 and a LC comprising the amino acid sequence of SEQ ID NO:50.

In other embodiments, provided antigen binding proteins include or are derived from one or more of the CDRs, variable heavy chains, variable light chains, heavy chains, and/or light chains of the following antibodies.

In another embodiment, the ABP comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences, wherein the VH CDR sequence is selected from the group consisting of (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 60; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 61; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 58 or SEQ ID NO: 62, and (b) a VL domain comprising at least one, at least two, or all three VLCDR sequences, wherein the VL CDR sequence is selected from the group consisting of (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 63 or SEQ ID NO: 68; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 64 or SEQ ID NO: 69; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 65 or SEQ ID NO: 70, with the proviso that in embodiments where the ABP comprises multiple CDRs, each CDR is selected from a different group.

In another embodiment, the ABP comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences, wherein the VH CDR sequence is selected from the group consisting of (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:56; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:57; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:58, and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences, wherein the VL CDR sequence is selected from the group consisting of (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:63; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO:64; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO:65, wherein the CDRs are defined by Kabat, and with the proviso that in embodiments where the ABP comprises multiple CDRs, each CDR is selected from a different group.

In another embodiment, the ABP comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences, wherein the VH CDR sequence is selected from the group consisting of (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:60; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:61; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:62, and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences, wherein the VL CDR sequence is selected from the group consisting of (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:68; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO:69; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO:70, wherein the CDRs are determined by IMGT, and with the proviso that in embodiments where the ABP comprises multiple CDRs, each CDR is selected from a different group.

In another embodiment, the ABP comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:56 or SEQ ID NO:60; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:57 or SEQ ID NO:61; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:58 or SEQ ID NO:62, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:63 or SEQ ID NO:68; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:64 or SEQ ID NO:69; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:65 or SEQ ID NO:70.

In another embodiment, the ABP comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:56; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:57; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:58, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:63; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:64; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:65; wherein the CDRs are defined by Kabat.

In another embodiment, the ABP comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:60; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:61; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:62, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:68; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:69; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:70; wherein the CDRs are determined by IMGT.

Certain ABPs comprise a VH comprising a CDR-H1, CDR-H2, and CDR-H3, wherein the CDRs of the VH have a total of at most 1, 2, 3, 4, or 5 amino acid changes relative to the corresponding CDR reference sequence, and wherein the CDR-H1 reference sequence has the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 60, the CDR-H2 reference sequence has the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 61, and the CDR-H3 reference sequence has the amino acid sequence of SEQ ID NO: 58 or SEQ ID NO: 62. In such embodiments, the amino acid changes are typically insertions, deletions, and/or substitutions. In some of these embodiments, the total number of amino acid changes is 1-3; in other embodiments, the total number of amino acid changes is 1 or 2. In certain of the foregoing embodiments, the changes are conservative amino acid substitutions.

In other embodiments, the ABP comprises a VL comprising CDR-L1, CDR-L2, and CDR-L3, wherein the CDRs of the VL have a total of at most 1, 2, 3, 4, or 5 amino acid changes relative to the corresponding CDR reference sequence, and wherein the CDR-L1 reference sequence has the amino acid sequence of SEQ ID NO: 63 or SEQ ID NO: 68, the CDR-L2 reference sequence has the amino acid sequence of SEQ ID NO: 64 or SEQ ID NO: 69, and the CDR-L3 reference sequence has the amino acid sequence of SEQ ID NO: 65 or SEQ ID NO: 70. In such embodiments, the amino acid changes are typically insertions, deletions, and/or substitutions. In some of these embodiments, the total number of amino acid changes is 1-3; in other embodiments, the total number of amino acid changes is 1 or 2. In certain of the foregoing embodiments, the changes are conservative amino acid substitutions.

In another embodiment, the ABP comprises (a) a VH comprising CDR-H1, CDR-H2, and CDR-H3, wherein the CDRs of the VH have a total of at most 1, 2, 3, 4, or 5 amino acid changes relative to the corresponding CDR reference sequence, and wherein the CDR-H1 reference sequence has the amino acid sequence of SEQ ID NO:56 or SEQ ID NO:60, the CDR-H2 reference sequence has the amino acid sequence of SEQ ID NO:57 or SEQ ID NO:61, and the CDR-H3 reference sequence has the amino acid sequence of SEQ ID NO:58 or SEQ ID NO:62, and (b) a VL comprising CDR-L1, CDR-L2, and CDR-L3, wherein the CDRs of the VL have a total of at most 1, 2, 3, 4, or 5 amino acid changes relative to the corresponding CDR reference sequence, and wherein the CDR-L1 reference sequence has the amino acid sequence of SEQ ID NO:63 or SEQ ID NO:68, the CDR-L2 reference sequence has the amino acid sequence of SEQ ID NO:64 or SEQ ID NO:69, and the CDR-L3 reference sequence has the amino acid sequence SEQ ID NO:65 or SEQ ID NO:70. In such embodiments, the amino acid changes are typically insertions, deletions and/or substitutions. In some of these embodiments, the total number of amino acid changes is 1-3; in other embodiments, the total number of amino acid changes is 1 or 2. In some of the foregoing embodiments, the changes are conservative amino acid substitutions.

In another embodiment, the ABP comprises a VH domain, wherein the VH domain sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with an amino acid sequence selected from any one of SEQ ID NOs: 9-16, provided that the ABP retains the ability to bind to ALPP and/or ALPPL2. In certain embodiments, such ABPs contain substitutions (e.g., conservative substitutions), insertions and/or deletions relative to a reference sequence (i.e., one of SEQ ID NOs: 9-16), provided that such ABPs retain the ability to bind to ALPP and/or ALPPL2. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in any one of SEQ ID NOs: 9-16 have been substituted, inserted and/or deleted. In some embodiments, 1-5 or 1-3 amino acids in the VH sequence have been substituted, inserted and/or deleted. In certain of these embodiments, such substitutions, insertions or deletions occur in regions outside the CDRs (ie, in the FRs).

In another embodiment, the ABP comprises a VL domain, wherein the VL domain sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with an amino acid sequence selected from any one of SEQ ID NOs: 22-33, provided that the ABP retains the ability to bind to ALPP and/or ALPPL2. In certain embodiments, such ABPs contain substitutions (e.g., conservative substitutions), insertions and/or deletions relative to a reference sequence (i.e., one of SEQ ID NOs: 22-33), provided that such ABPs retain the ability to bind to ALPP and/or ALPPL2. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in any one of SEQ ID NOs: 22-33 have been substituted, inserted and/or deleted. In some embodiments, 1-5 or 1-3 amino acids in the VL sequence have been substituted, inserted and/or deleted. In certain of these embodiments, such substitutions, insertions or deletions occur in regions outside the CDRs (ie, in the FRs).

In another embodiment, the ABP comprises (a) a VH domain, wherein the VH domain sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from any one of SEQ ID NOs: 9-16; and (b) a VL domain, wherein the VL domain sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from any one of SEQ ID NOs: 22-33, provided that the ABP retains the ability to bind ALPP and/or ALPPL2.

In another embodiment, the ABP comprises (a) a VH domain, wherein the VH domain sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:15; and (b) a VL domain, wherein the VL domain sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:30, provided that the ABP retains the ability to bind ALPP and/or ALPPL2.

The antigen binding protein in any of the foregoing embodiments can be any form of antibody. Thus, the antigen binding protein described in any of the above embodiments can be, for example, a monoclonal antibody, a multispecific antibody, a human antibody, a humanized antibody, or a chimeric antibody, and any of the above ALPP binding fragments, such as a single chain antibody, a Fab fragment, a F(ab') fragment, or a fragment produced by a Fab expression library. The antibody can be any immunoglobulin isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass.

In certain embodiments, the ABP having the CDR and/or variable domain sequences described herein is an antigen binding fragment (e.g., a human antigen binding fragment), and includes, but is not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and fragments comprising a VL or VH domain. Antigen binding fragments, including single-chain antibodies, may comprise one or more variable regions alone or in combination with all or part of the following: hinge region, CH1, CH2, CH3, and CL domains. The present disclosure also includes antigen binding fragments comprising any combination of one or more variable regions and hinge region, CH1, CH2, CH3, and CL domains.

ABPs can be monospecific, bispecific, trispecific, or have greater multispecificity. Multispecific antibodies can be specific for different epitopes of ALPP and/or ALPPL2, or can be specific for both ALPP and/or ALPPL2 and a heterologous protein. See, e.g., PCT Publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., 1991, J. Immunol. 147:60 69; U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; and Kostelny et al., 1992, J. Immunol. 148:1547 1553.

In any of the embodiments described herein, one or several amino acids (e.g., 1, 2, 3, or 4) at the amino or carboxyl terminus of the light and/or heavy chain, such as the C-terminal lysine of the heavy chain, can be deleted or derivatized in some or all of the molecules in the composition. A specific example of such a modification is an ABP in which the carboxyl terminal lysine of the heavy chain is deleted (e.g., as part of a post-translational modification). In addition, it is understood that any sequence described herein includes post-translational modifications of the particular sequence during expression of the ABP in cell culture (e.g., CHO cell culture).

B. Chimeric Antigen Binding Proteins

In certain embodiments, the antigen-binding proteins provided herein are chimeric antibodies. In some embodiments, chimeric antibodies comprise non-human variable regions (e.g., variable regions derived from mice, rats, hamsters, rabbits, or non-human primates such as monkeys) and human constant regions. In another example, chimeric antibodies are "class switched" antibodies, in which the class or subclass has been changed from the class or subclass of the parent antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). Chimeric antibodies include antigen-binding fragments thereof.

Non-limiting exemplary chimeric antibodies include chimeric antibodies comprising any heavy chain and/or light chain variable regions described herein. In certain embodiments, the heavy chain and/or light chain variable domains are selected from, and other non-limiting exemplary chimeric antibodies include chimeric antibodies comprising heavy chain HVR sequences (e.g., CDRs) or portions thereof and/or light chain HVR sequences (e.g., CDRs) as provided herein.

C. Humanized Antigen Binding Protein

In certain embodiments, the ABP is a humanized antibody that binds to ALPP and/or ALPPL2. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Humanized antibodies are genetically engineered antibodies in which HVRs (e.g., CDRs) or portions thereof from non-human "donor" antibodies are transplanted into human "acceptor" antibody sequences (see, e.g., Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,205; and Foote, US 6,881,557).

The acceptor antibody sequence can be, for example, a mature human antibody sequence, a complex of such a sequence, a consensus sequence of a human antibody sequence, or a germline region sequence. A human acceptor sequence with a high degree of sequence identity in the variable region framework can be selected to match the canonical form and other standards between the acceptor and the donor HVR or CDR. Therefore, a humanized antibody is an antibody with a variable region framework sequence and a constant region (if present) completely or substantially from the HVR or CDR of a donor antibody and completely or substantially from a human antibody sequence. Similarly, a humanized heavy chain generally has all three HVRs or CDRs completely or substantially from a donor antibody heavy chain, and a heavy chain variable region framework sequence and a heavy chain constant region (if present) substantially from a human heavy chain variable region framework and a constant region sequence. Similarly, a humanized light chain generally has all three CDRs completely or substantially from a donor antibody light chain, and a light chain variable region framework sequence and a light chain constant region (if present) substantially from a human light chain variable region framework and a constant region sequence. When at least 80%, 85%, 90%, 95% or 100% of the corresponding residues (as defined by Kabat) are identical between the corresponding HVRs or CDRs, the HVRs or CDRs in the humanized antibody are substantially derived from the corresponding HVRs or CDRs in the non-human antibody. When at least 80%, 85%, 90%, 95% or 100% of the corresponding residues as defined by Kabat are identical, the variable region framework sequence of the antibody chain or the constant region of the antibody chain is substantially derived from the human variable region framework sequence or the human constant region, respectively.

Although humanized antibodies typically incorporate all six HVRs (e.g., CDRs, preferably as defined by Kabat) from a mouse antibody, they can also be made having fewer than all (e.g., at least 3, 4, or 5) HVRs or CDRs from a mouse antibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320:415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; and Tamura et al., Journal of Immunology, 164:1432-1441, 2000).

Certain amino acids from human variable region framework residues can be selected for substitution based on their possible effects on HVR (e.g., CDR) conformation and/or binding to antigen. Such possible effects are studied by modeling, examining the characteristics of amino acids at specific positions, or empirically observing the effects of substitution or mutagenesis of specific amino acids.

For example, when there is an amino acid difference between a murine framework residue in a murine variable region and a selected human variable region framework residue, the human framework amino acid can be substituted with the equivalent framework amino acid from a mouse antibody, where it is reasonably expected that the amino acid:

(1) Non-covalent direct binding to antigens,

(2) adjacent to the HVR or CDR region,

(3) otherwise interact with the HVR or CDR region (e.g., Inside);

(4) mediate the interaction between heavy and light chains, or

(5) is the result of somatic mutations in the mouse chain.

(6) is the glycosylation site.

Framework residues of classes (1)-(3) are sometimes referred to interchangeably as canonical residues and vernier residues. Canonical residues refer to framework residues that define the canonical class of donor CDR loops that determine the conformation of the CDR loops (Chothia and Lesk, J. Mol. Biol. 196, 901-917 (1987); Thornton and Martin, J. Mol. Biol., 263, 800-815, 1996). Vernier residues refer to a layer of framework residues that supports the conformation of the antigen-binding loop and plays a role in fine-tuning the coordination of the antibody with the antigen (Foote and Winter, 1992, J Mol Bio. 224, 487-499).

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

Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al. (1993) J. Immunol. 151:2296); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; and Presta et al. (1993) J. Immunol, 151:26 23); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633); and framework regions from screening FR libraries (see, e.g., Baca et al., (1997) J. Biol. Chem. 272: 10678-10684 and Rosok et al., (1996) J. Biol. Chem. 271: 22611-22618).

Non-limiting exemplary humanized antibodies include humanized antibodies comprising or derived from any CDR and/or heavy chain and/or light chain variable region disclosed herein. Specific examples of such antibodies include humanized forms of mouse antibody 12F3. Such humanized variants of mouse antibody 12F3 are named HGLF, which comprises: a mature heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a mature light chain variable region comprising the amino acid sequence of SEQ ID NO: 30. The humanized antibodies of the present invention include variants of the HGLF humanized antibody, wherein the mature variable region of the humanized heavy chain shows at least 90%, 95% or 99% identity with SEQ ID NO: 15, and the mature variable region of the humanized light chain shows at least 90%, 95% or 99% sequence identity with SEQ ID NO: 30. Preferably, in such antibodies, some or all of the back mutations in HGLF are retained. In other words, at least 1, 2, 3, 4, 5, 6 or preferably all 7 of the heavy chain positions H30, H37, H48, H49, H73, H78 and H93 are occupied by T, V, L, A, N, L and A, respectively. Likewise, at least 1, 2, 3, 4 or preferably all 4 of the light chain positions L2, L38, L49 and L69 are occupied by T, Y, H and R, respectively. HGLF is described in more detail in the Examples and has the sequence shown in Figures 5 to 8.

D. Exemplary Antibody Constant Regions

For those embodiments in which the ABP is an antibody, the heavy chain and light chain variable regions of the antibodies described herein can be connected to at least a portion of a human constant region. In some embodiments, the human heavy chain constant region is an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region has an isotype selected from κ and λ. In some embodiments, the antibodies described herein comprise a human IgG constant region. In some embodiments, the antibodies described herein comprise a human IgG4 heavy chain constant region. In some of these embodiments, the antibodies described herein comprise an S228P mutation in a human IgG4 constant region. In some embodiments, the antibodies described herein comprise a human IgG4 constant region and a human κ light chain.

In the present specification and claims, unless explicitly stated or known to those skilled in the art, the numbering of the residues in the immunoglobulin heavy chain is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991), which is expressly incorporated herein by reference. The "EU index in Kabat" refers to the residue numbering of the human IgG1EU antibody.

Human constant regions show allotypic variation and homologous allotypic variation between different individuals, i.e., the constant region may be different in one or more polymorphic positions in different individuals. Homologous allotypes differ from allotypes in that serum that recognizes homologous allotypes binds to non-polymorphic regions of one or more other isotypes. Reference to human constant regions includes constant regions having any natural allotype or any arrangement of residues occupying polymorphic positions in natural allotypes. In addition, there may be up to 1, 2, 5 or 10 mutations relative to the natural human constant region, such as those noted above, to reduce Fcγ receptor binding or increase binding to FcRn.

In some embodiments, one or more amino acids at the amino or carboxyl terminus of a light and/or heavy chain, such as the C-terminal lysine of a heavy chain, may be deleted or derivatized in part or all of the molecule.

The choice of constant region depends in part on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, and/or complement-dependent cytotoxicity are desired. For example, human isotypes IgG1 and IgG3 have strong complement-dependent cytotoxicity, human isotype IgG2 has weak complement-dependent cytotoxicity, and human IgG4 lacks complement-dependent cytotoxicity. Human IgG1 and IgG3 also induce stronger cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region can be λ or κ.

In addition, as described in more detail below, substitutions can be made in the constant region to reduce or increase effector function, such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to increase half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004).

E. Variants

Antigen-binding proteins provided herein also include amino acid sequence variants of antigen-binding proteins provided herein.For example, variants with improved antibody binding affinity and/or other biological properties can be prepared.The amino acid sequence variants of antigen-binding proteins can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antigen-binding proteins or by peptide synthesis.Such modifications include, for example, the disappearance and/or insertion and/or substitution of residues in the amino acid sequence of the antigen-binding proteins.Any combination of disappearance, insertion and substitution can be carried out to obtain the final construct, and the precursor condition is that the final construct has the desired characteristics, for example, antigen binding.

1. Substitution, insertion and deletion variants

In some embodiments, the antigen binding protein is a variant having one or more amino acid substitutions, deletions and/or insertions relative to the antigen binding proteins described herein. In certain such embodiments, the variant has one or more amino acid substitutions. In other such embodiments, the substitution is a conservative amino acid substitution.

Amino acid substitutions may include, but are not limited to, replacement of one amino acid in a polypeptide with another amino acid. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. Naturally occurring residues can be divided into several categories based on common side chain properties:

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

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

(3) Acidic: Asp, Glu;

(4) Basic: His, Lys, Arg;

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

(6) Aromatic: Trp, Tyr, Phe.

The target site of substitution mutagenesis includes CDR and FR.Conservative substitutions are shown in the following table 2 under the heading "preferred substitutions".More substantial changes are provided under the heading "exemplary substitutions" in Table 2, and are further described below about amino acid side chain categories.Amino acid substitutions can be introduced into the target antibody, and the product is screened for desired activity, such as retained/improved antigen binding, reduced immunogenicity or improved ADCC or CDC.

Table 2

Original residue Exemplary Substitutions Preferred substitution Ala Val; Leu; Ile Val Arg Lys; Gln; Asn Lys Asn Gln; His; Asp; Lys; Arg Gln Asp Glu; Asn Glu Cys Ser; Ala Ser Gln Asn；Glu Asn Glu Asp; Gln Asp Gly Pro; Ala Ala His Asn; Gln; Lys; Arg Arg Ile Leu; Val; Met; Ala; Phe; Norleucine Leu Leu Norleucine; Ile; Val; Met; Ala; Phe Ile Lys Arg; Gln; Asn Arg Met Leu; Phe; Ile Leu Phe Trp; Leu; Val; Ile; Ala; Tyr Leu Pro Ala Ala Ser Thr；Ala；Cys Thr Thr Val; Ser Ser Trp Tyr; Phe Tyr Tyr Trp; Phe; Thr; Ser Phe Val Ile; Leu; Met; Phe; Ala; norleucine Leu

Non-conservative substitutions involve exchanging a member of one of these classes for a member of another class.

When changing the amino acid sequence of an antigen binding protein (e.g., an anti-ALPP/ALPPL2 antibody), in some embodiments, the hydropathic index of the amino acids can be considered. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics, as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic amino acid index in conferring interactive biological function on proteins is known in the art. Kyte et al., 1982, J. Mol. Biol., 157: 105-131. It is known that certain amino acids can be substituted with other amino acids having a similar hydropathic index or score and still retain similar biological activity. When making changes based on the hydropathic index, in certain embodiments, substitutions of amino acids with a hydropathic index within ±2 are included. In certain embodiments, those amino acids within ±1 are included, and in certain embodiments, those amino acids within ±0.5 are included.

It is also understood in the art that substitution of similar amino acids can be effectively made on the basis of hydrophilicity, particularly when the biologically functional protein or peptide (e.g., antibody) so produced is intended for use in immunological embodiments, as in the case of the present invention. In certain embodiments, the greatest local average hydrophilicity of a protein (as influenced by the hydrophilicity of its neighboring amino acids) correlates with its immunogenicity and antigenicity, i.e., with the biological properties of the protein.

These amino acid residues have been assigned the following hydrophilicity values: arginine (+3.0); lysine (+3.0 ± 1); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). When making changes based on similar hydrophilicity values, in certain embodiments, substitution of amino acids with hydrophilicity values within ±2 is included, in certain embodiments, those within ±1 are included, and in certain embodiments, those within ±0.5 are included. Epitopes can also be identified from the primary amino acid sequence based on hydrophilicity. These regions are also referred to as "epitope core regions."

Changes (e.g., substitutions) can be made in the CDRs, for example to improve antibody affinity. Such changes can be made in CDR "hot spots", i.e., residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)) and/or residues that contact the antigen, and the resulting variant VH or VL binding affinity is tested. Affinity maturation by constructing a secondary library and reselecting from the secondary library is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or mutagenesis for oligonucleotides). Then, a secondary library is created. Then, the library is screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves a method for CDRs, wherein multiple CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

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

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

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N- or C-termini of antibodies to enzymes (e.g., for ADEPT) or polypeptides that extend the serum half-life of the antibody.

2. Variants with modified Fc regions

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

In certain embodiments, antibody variants with improved or reduced binding to FcRs are prepared. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)). In some embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and/or 334 (EU numbering of residues) of the Fc region. For example, systematic substitution of solvent-exposed amino acids in the human IgG1 Fc region produced IgG variants with altered FcγR binding affinity (Shields et al., 2001, J. Biol. Chem. 276:6591-604). A subset of these variants involving substitutions to Ala at Thr256/Ser298, Ser298/Glu333, Ser298/Lys334, or Ser298/Glu333/Lys334 demonstrated increased binding affinity to FcγRs and ADCC activity when compared to the parental IgG1 (Shields et al., 2001, J. Biol. Chem. 276:6591-604; Okazaki et al., 2004, J. Mol. Biol. 336:1239-49).

In some embodiments, changes are made in the Fc region to alter (i.e., improve or reduce) Clq binding and/or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000). For example, the complement binding activity (Clq binding and CDC activity) of an antibody can be improved by substitutions at Lys326 and Glu333 (Idusogie et al., 2001, J. Immunol. 166: 2571-2575). The same substitutions on the human IgG2 backbone can convert an antibody isotype that binds poorly to Clq and is severely deficient in complement activation activity to an antibody isotype that can both bind to Clq and mediate CDC (Idusogie et al., 2001, J. Immunol. 166: 2571-75). Several other methods have also been applied to improve the complement binding activity of antibodies. For example, grafting the 18 amino acid carboxyl terminal tail of IgM to the carboxyl terminal of IgG greatly enhances their CDC activity. This is observed even for IgG4, which usually has no detectable CDC activity (Smith et al., 1995, J. Immunol. 154: 2226-36). In addition, replacing Ser444 near the carboxyl terminal of the IgG1 heavy chain with Cys induces tail-to-tail dimerization of IgG1, and its CDC activity is 200 times that of monomeric IgG1 (Shopes et al., 1992, J. Immunol. 148: 2918-22). In addition, bispecific double antibody constructs specific for C1q also confer CDC activity (Kontermann et al., 1997, Nat. Biotech. 15: 629-31).

Complement activity can be reduced by mutating at least one of the amino acid residues 318, 320, and 322 of the heavy chain to a residue with a different side chain, such as Ala. Other alkyl-substituted nonionic residues, such as Gly, Ile, Leu, or Val, or aromatic nonpolar residues such as Phe, Tyr, Trp, and Pro, replacing any of the three residues can also reduce or eliminate C1q binding. Ser, Thr, Cys, and Met can be used for residues 320 and 322, but not 318, to reduce or eliminate C1q binding activity. Replacing residue 318 (Glu) with a polar residue can alter but not eliminate C1q binding activity. Replacing residue 297 (Asn) with Ala results in elimination of lytic activity, but only a slight decrease in affinity for C1q (about one-third of the original). This change destroys the glycosylation site and the presence of the carbohydrate required for complement activation. Any other substitution at this site will also destroy the glycosylation site. The following mutations and any combination thereof also reduced CIq binding: D270A, K322A, P329A and P311S (see WO 06/036291).

The half-life of the antibodies provided herein can be increased or decreased to change their therapeutic activity. FcRn is a receptor structurally similar to the MHC class I antigens that are non-covalently bound to β2-microglobulin. FcRn regulates the catabolism of IgG and their transcytosis across tissues (Ghetie and Ward, 2000, Annu. Rev. Immunol. 18: 739-766; Ghetie and Ward, 2002, Immunol. Res. 25: 97-113). IgG-FcRn interaction occurs at pH 6.0 (pH of intracellular vesicles), but does not occur at pH 7.4 (pH of blood); This interaction enables IgG to recirculate back into the circulation (Ghetie and Ward, 2000, Ann. Rev. Immunol. 18: 739-766; Ghetie and Ward, 2002, Immunol. Res. 25: 97-113). The regions on human _IgG1 involved in FcRn binding have been mapped (Shields et al., 2001, J. Biol. Chem. 276: 6591-604). Alanine substitutions at positions Pro238, Thr256, Thr307, Gln311, Asp312, Glu380, Glu382, or Asn434 of human IgG1 enhance FcRn binding (Shields et al., 2001, J. Biol. Chem. 276: 6591-604). IgG1 molecules with these substitutions have longer serum half-lives. Therefore, these modified _IgG1 molecules may be able to perform their effector functions for a longer period of time compared to unmodified _IgG1 , and thus exert their therapeutic efficacy. Other exemplary substitutions for increasing binding to FcRn include Gln at position 250 and/or Leu at position 428. Other studies have shown that the binding of the Fc region to FcRn can be improved by introducing one or more substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, such as substitution of Fc region residue 434 (see, e.g., U.S. Pat. Nos. 7,371,826 and 7,361,740).

3. Antibody variants with modified glycosylation

In certain embodiments, the antibodies provided herein include one or more modifications to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites of antibodies can be conveniently achieved by changing the amino acid sequence to create or remove one or more glycosylation sites.

In the case where the antibody comprises an Fc region, the carbohydrate connected thereto can be changed. The natural antibody produced by mammalian cells generally comprises branched, biantennary oligosaccharides, which are generally connected to the Asn297 of the CH2 domain in the Fc region by an N-key. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose connected to GlcNAc in the "stem" of the biantennary oligosaccharide structure.

Engineering this glycoform on IgG can significantly improve IgG-mediated ADCC. Adding a bisected N-acetylglucosamine modification to the glycoform (Umana et al., 1999, Nat. Biotechnol. 17: 176-180; Davies et al., 2001, Biotech. Bioeng. 74: 288-94) or removing fucose from the glycoform (Shields et al., 2002, J. Biol. Chem. 277: 26733-40; Shinkawa et al., 2003, J. Biol. Chem. 278: 6591-604; Niwa et al., 2004, Cancer Res. 64: 2127-33) are two examples of IgG Fc engineering, which improves the binding between IgG Fc and FcγR, thereby enhancing Ig-mediated ADCC activity. Antibodies including such substitutions or engineering are included in some embodiments provided herein.

In certain embodiments, antibodies are provided having a carbohydrate structure lacking fucose (directly or indirectly) attached to an Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all sugar structures (e.g., complexes, hybrids, and high mannose structures) attached to Asn 297, as measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008/077546. Asn297 refers to an asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, e.g., U.S. Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US2003/0157108; WO 2000/61739; WO 2001/29246; US2003/0115614; US2002/0164328; US2004/0093621; US2004/0132140; US2004/0110704; US2004/0110282; US2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); U.S. Patent Application No. US2003/0157108A1, Presta, L; and WO 2004/056312 A1, Adams et al., particularly in Example 11) and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4): 680-688 (2006); and WO2003/085107).

Other antibodies containing bisected oligosaccharides are also provided, for example, biantennary oligosaccharides connected to the Fc region of the antibody are bisected by GlcNAc. Such antibodies may have reduced fucosylation and/or improved ADCC function. Examples of such antibodies are described in, for example, WO 2003/011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.) and US2005/0123546 (Umana et al.). Antibodies having at least one galactose residue in the oligosaccharide connected to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described in, for example, WO 1997/30087 (Patel et al.), WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.).

4. Cysteine engineered antibody variants

In some embodiments, the antibody variants provided herein include replacing natural amino acids with cysteine residues at amino acid positions 234, 235, 237, 239, 267, 298, 299, 326, 330 or 332 in human IgG1 isotypes, preferably S239C mutations (substitution of constant regions is according to EU index). The presence of additional cysteine residues allows the formation of interchain disulfide bonds. The formation of such interchain disulfide bonds can cause steric hindrance, thereby reducing the affinity of the Fc region-FcγR binding interaction. The cysteine residues introduced in or near the Fc region of the IgG constant region can also be used as sites for conjugation with therapeutic agents (e.g., using thiol-specific reagents to couple cytotoxic drugs, such as maleimide derivatives of drugs). The presence of therapeutic agents causes steric hindrance, thereby further reducing the affinity of the Fc region-FcγR binding interaction. Other substitutions at any of positions 234, 235, 236 and/or 237 reduce affinity for Fcγ receptors, particularly the FcγRI receptor (see, e.g., US 6,624,821, US 5,624,821).

In other cysteine engineered antibody variants, one or more reactive thiol groups are located at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to produce immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and 5400 (EU numbering) of the heavy chain Fc region. The production of cysteine engineered antibodies is described, for example, in U.S. Patent No. 7,521,541.

5. Exemplary Fc variants

Certain ABPs provided include the following modifications to the constant region.

F. Competitive antigen binding protein

The antigen binding proteins provided herein include those that compete with one of the above exemplary ABPs or fragments for specific binding to ALPP and/or ALPPL2 (e.g., human ALPP of SEQ ID NO: 2 and/or human ALPPL2 of SEQ ID NO: 4). In some of these embodiments, the test and reference ABPs cross-compete with each other. Such ABPs may bind to the same epitope as one of the antigen binding proteins described herein, or bind to overlapping epitopes. In one embodiment, such ABPs bind to an epitope having an amino acid sequence of SEQ ID NO: 73 and/or SEQ ID NO: 74. It is expected that ABPs including fragments that compete with the exemplary ABPs exhibit similar functional properties (e.g., one or more of the above activities).

In some embodiments, the ABPs provided include those that compete with antibodies having: (a) all six CDRs listed for the same antibody as in SEQ ID NOs:56-58 or 60-62 and 63-65 or 68-70; (b) the VH and VL listed for the same antibody as in SEQ ID NOs:15 and 30; or (c) the specified light and heavy chains for the same antibody as in SEQ ID NOs:40 and 50.

G. Antigen Binding Proteins Binding to the Same Epitope

In another embodiment, the antigen binding proteins provided include those that bind to the same epitope as any ABP described herein. A variety of techniques can be used to identify ABPs that bind to the same epitope as one or more ABPs described herein. Such methods include, for example, competition assays described herein, screening of peptide fragments, protein footprints based on MS, alanine or glutamine scanning methods, and x-ray analysis of crystals of antigen: antigen binding protein complexes that provide atomic resolution of the epitope.

One method for determining an epitope or epitope region to which a specific antibody binds (an "epitope region" is a region that contains or overlaps with an epitope) involves evaluating the binding of an ABP to a peptide comprising an ALPP and/or ALPPL2 fragment (e.g., a non-denatured or denatured fragment). A series of overlapping peptides comprising the sequence of ALPP and/or ALPPL2 (e.g., human ALPP and/or human ALPPL2) can be prepared and screened for binding, for example in a direct ELISA, a competitive ELISA (in which the peptides are evaluated for their ability to prevent the binding of an antibody to ALPP and/or ALPPL2 bound to the wells of a microtiter plate), or on a chip. Such peptide screening methods may not be able to detect some discontinuous functional epitopes, i.e., functional epitopes involving amino acid residues that are discontinuous along the primary sequence of the ALPP and/or ALPPL2 polypeptide chain.

In other embodiments, regions containing residues that are in contact with or buried by the antibody can be identified by mutating specific residues in ALPP and/or ALPPL2 and determining whether the ABP can bind to the mutated or variant ALPP and/or ALPPL2 protein. By making multiple individual mutations, residues that play a direct role in binding or are close enough to the antibody can be identified so that the mutation can affect the binding between the antigen binding protein and the antigen. Based on the knowledge of these amino acids, the domain or region of the antigen containing the residues that are in contact with the ABP or covered by the antibody can be elucidated. Such a domain may include the binding epitope of the ABP. The general method of this scanning technology involves replacing amino acids in the wild-type polypeptide with arginine and/or glutamic acid residues (usually alone). These two amino acids are commonly used in such scanning technologies because they are charged and bulky, and therefore have the potential to destroy the binding between the ABP and ALPP and/or ALPPL2 in the ALPP and/or ALPPL2 region into which the mutation is introduced. The arginine present in the wild-type antigen is replaced by glutamic acid. A variety of such individual mutants are obtained and the collected binding results are analyzed to determine which residues affect binding (see, e.g., Nanevicz, T. et al., 1995, J. Biol. Chem., 270:37, 21619-21625 and Zupnick, A. et al., 2006, J. Biol. Chem., 281:29, 20464-20473).

Another method for identifying epitopes is MS-based protein footprinting, such as hydrogen/deuterium exchange mass spectrometry (HDX-MS) and fast photochemical oxidation of proteins (FPOP). Methods for performing HDX-MS are described, for example, in Wei et al. (2014) Drug Discovery Today 19:95. Methods for performing FPOP are described, for example, in Hambley and Gross (2005) J. American Soc. Mass Spectrometry 16:2057.

The epitope to which the ABP binds can also be determined by structural methods such as X-ray crystallography, molecular modeling, and nuclear magnetic resonance (NMR) spectroscopy, including NMR determination of the H-D exchange rates of labile amide hydrogens in the antigen when free and when bound in a complex with the ABP (see, e.g., Zinn-Justin et al. (1992) Biochemistry 31, 11335-11347; and Zinn-Justin et al. (1993) Biochemistry 32, 6884-6891).

X-ray crystallographic analysis can be performed using any method known in the art. Examples of crystallization methods are described, for example, by Giege et al. (1994) Acta Crystallogr. D50: 339-350 and McPherson (1990) Eur. J. Biochem. 189: 1-23. Such crystallization methods include micro-batch (e.g., Chayen (1997) Structure 5: 1269-1274), hanging drop vapor diffusion (e.g., McPherson (1976) J. Biol. Chem. 251: 6300-6303), inoculation, and dialysis. Once formed, the ABP:antigen crystals themselves can be studied using well-known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, published by Molecular Simulations, Inc.; see, e.g., Blundell and Johnson (1985) Meth. Enzymol. 114 and 115, H. W. Wyckoff et al., eds., Academic Press; U.S. Patent Application Publication No. 2004/0014194) and BUSTER (Bricogne (1993) Acta Cryst. D 49:37-60; Bricogne (1997) Meth. Enzymol. 276A:361-423, Carter and Sweet, eds.; Roversi et al. (2000) Acta Cryst. D 56:1313-1323).

In some embodiments, the ABP binds to a contiguous epitope. In a preferred embodiment, the ABP binds to an epitope having an amino acid sequence of SEQ ID NO:73 and/or SEQ ID NO:74.

H. Other Exemplary Forms

An antigen binding protein (e.g., an antibody or antigen binding fragment thereof) can be a single polypeptide, or can comprise two, three, four, five, six, seven, eight, nine, or ten (same or different) polypeptides. In some embodiments where the antibody or antigen binding fragment thereof is a single polypeptide, the antibody or antigen binding fragment can comprise a single antigen binding domain or two antigen binding domains. In some embodiments where the antibody or antigen binding fragment is a single polypeptide and comprises two antigen binding domains, the first antigen binding domain and the second antigen binding domain can be the same or different from each other (and can specifically bind to the same or different antigens or epitopes).

Different portions of the antigen binding proteins described herein can be arranged in various configurations to obtain additional antigen binding proteins. For example, in some embodiments where the antibody or antigen binding fragment is a single polypeptide, the first antigen binding domain and the second antigen binding domain (if present) can each be independently selected from: a VH domain, a VHH domain, a VNAR domain, and a scFv. In some embodiments where the antibody or antigen binding fragment is a single polypeptide, the antibody or antigen binding fragment can be (scFv) ₂ , nanobody, nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH-CL-scFv, HSAbody, scDiabody-HAS, tandem-scFv, Adnectin, DARPin, fibronectin and DEP conjugates. Other examples of antigen binding domains that can be used when the antibody or antigen binding fragment is a single polypeptide are known in the art.

A _VHH domain is a single monomeric variable antibody domain that can be found in camelids. _{A VNAR} domain is a single monomeric variable antibody domain that can be found in cartilaginous fish. Non-limiting aspects of _VHH domains and _VNAR domains are described, for example, in Cromie et al., Curr. Top. Med. Chem. 15:2543-2557, 2016; De Genst et al., Dev. Comp. Immunol. 30:187-198, 2006; De Meyer et al., Trends Biotechnol. 32:263-270, 2014; Kijanka et al., Nanomedicine 10:161-174, 2015; Kovaleva et al., Expert. Opin. Biol. Ther. 14:1527-1539, 2014; Krah et al., Immunopharmacol. Immunotoxicol. 38:21-28, 2016; Mujic-Delic et al., Trends Biotechnol. Pharmacol. Sci. 35:247-255, 2014; Muyldermans, J. Biotechnol. 74:277-302, 2001; Muyldermans et al., Trends Biochem. Sci. 26:230-235, 2001; Muyldermans, Ann. Rev. Biochem. 82:775-797, 2013; Rahbarizadeh et al., Immunol. Invest. 40:299-338, 2011; Van Audenhove et al., EBioMedicine 8:40-48, 2016; Van Bockstaele et al., Curr. Opin. Investig. Drugs 10:1212-1224, 2009; Vincke et al., Methods Mol. Biol. 911: 15-26, 2012; and Wesolowski et al., Med. Microbiol. Immunol. 198: 157-174, 2009.

In some embodiments where the antibody or antigen binding fragment is a single polypeptide and comprises two antigen binding domains, the first antigen binding domain and the second antigen binding domain may both be VHH domains, or at least one antigen binding domain may be a VHH domain. In some embodiments where the antibody or antigen binding fragment is a single polypeptide and comprises two antigen binding domains, the first antigen binding domain and the second antigen binding domain are both V _NAR domains, or at least one antigen binding domain is a V _NAR domain. In some embodiments where the antibody or antigen binding domain is a single polypeptide, the first antigen binding domain is a scFv domain. In some embodiments where the antibody or antigen binding fragment is a single polypeptide and comprises two antigen binding domains, the first antigen binding domain and the second antigen binding domain may both be scFv domains, or at least one antigen binding domain may be a scFv domain.

In some embodiments, the antibody or antigen-binding fragment may comprise two or more polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten polypeptides). In some embodiments where the antibody or antigen-binding fragment comprises two or more polypeptides, two, three, four, five, or six of the two or more polypeptides may be identical.

In some embodiments where the antibody or antigen-binding fragment comprises two or more polypeptides (e.g., two, three, four, five, six, seven, eight, nine, or ten polypeptides), the two or more polypeptides of the antibody or antigen-binding fragment can be assembled (e.g., non-covalently assembled) to form one or more antigen-binding domains, such as an antigen-binding fragment of an antibody (e.g., any antigen-binding fragment of an antibody described herein), VHH-scAb, VHH-Fab, bi-scFab, F(ab') ₂ , diabody, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knob-in-hole common light chain, knob-in-hole assembly, charge pair, Fab-arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, diabody-CH3, triplet, minibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab') ₂ -scFv ₂ , scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, VHH-Fc, tandem VHH-Fc, VHH-Fc KiH, Fab-VHH-Fc, Intrabody, docking-locking, ImmTAC, IgG-IgG conjugates, Cov-X-Body, scFv1-PEG-scFv2, Adnectin, DARPin, fibronectin and DEP conjugates. See, e.g., Spiess et al., Mol. Immunol. 67:95-106, 2015, which is incorporated herein by reference in its entirety, for a description of these elements.

In some embodiments, the antigen binding protein is based on a non-immunoglobulin scaffold. Examples of other scaffolds into which binding domains such as those described herein (e.g., HVRs or CDRs) can be inserted or grafted include, but are not limited to, human fibronectin (e.g., the 10th extracellular domain of human fibronectin III), neocarcinostatisin CBM4-2, anti-catenin derived from lipocalin, designed ankyrin repeat domains (DARPins), protein-A domains (protein Z), Kunitz domains, Im9, TPR proteins, zinc finger domains, pVIII, GC4, transferrin, the B domain of SPA, Sac7d, the A-domain, the SH3 domain of Fyn kinase, and C-type lectin-like domains (see, e.g., Gebauer and Skerra (2009) Curr. Opin. Chem. Biol., 13:245-255; Binz et al. (2005) Nat. Biotech. 23:1257-1268; and Yu et al. (2017) Annu Rev Anal Chem 10:293-320, each of which is incorporated herein by reference in its entirety).

IV. Antigen Binding Protein Expression and Production

A. Nucleic Acid Molecules Encoding Antigen Binding Proteins

Also provided are nucleic acid molecules encoding antigen-binding proteins or portions thereof as described herein. Such nucleic acids include, for example: 1) those encoding antigen-binding proteins (e.g., antibodies or fragments thereof) or derivatives or variants thereof; 2) polynucleotides encoding heavy and/or light chains, VH and/or VL domains, or one or more HVRs or CDRs (e.g., 1, 2, or all 3 VH HVRs or CDRs or 1, 2, or all 3 VL HVRs or CDRs) within the variable domains; 3) polynucleotides sufficient to be used as hybridization probes, PCR primers, or sequencing primers to identify, analyze, mutate, or amplify such encoding polynucleotides; 4) antisense nucleic acids for inhibiting the expression of such encoding polynucleotides; and 5) the aforementioned complementary sequences. Nucleic acids can be of any length. They 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, or 1,000 or more nucleotides in length, and/or can comprise one or more additional sequences, such as regulatory sequences, and/or be part of a larger nucleic acid, such as a vector. The nucleic acids can be single-stranded or double-stranded.

Nucleic acid molecules can be present in intact cells, cell lysates or in partially purified or substantially pure forms. When purified from other cellular components or other contaminants such as other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA connected to naturally isolated DNA) or proteins by standard techniques (including alkali/SDS treatment, CsCl bands, column chromatography, restriction enzymes, agarose gel electrophoresis and other techniques well known in the art), nucleic acids are "isolated" or "substantially pure". See F.Ausubel et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. Nucleic acid as described herein can be, for example, DNA or RNA, and can contain or not contain intron sequences. In certain embodiments, nucleic acid is a cDNA molecule.

In one embodiment, the nucleic acid molecule encoding the VH sequence of the antibody provided herein comprises SEQ ID NO: 71. In another embodiment, the nucleic acid molecule encoding the VL sequence of the antibody provided herein comprises SEQ ID NO: 72. In another embodiment, the nucleic acid molecule encoding the VH and VL sequences of the antibody provided herein comprises SEQ ID NO: 71 and SEQ ID NO: 72, respectively.

Thus, nucleic acid molecules comprising polynucleotides encoding one or more chains of ABP, such as anti-ALPP/ALPPL2 antibodies, are provided. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding a heavy chain or a light chain of an ABP (e.g., an anti-ALPP/ALPPL2 antibody). In some embodiments, the nucleic acid molecule comprises both a polynucleotide sequence encoding the heavy chain of an ABP (e.g., an anti-ALPP/ALPPL2 antibody) and a polynucleotide sequence encoding its light chain. In some embodiments, the first nucleic acid molecule comprises a first polynucleotide sequence encoding a heavy chain, and the second nucleic acid molecule comprises a second polynucleotide sequence encoding a light chain.

In one embodiment, the nucleic acid molecule comprises a polynucleotide encoding the VH of one of the antibodies provided herein. In another embodiment, the nucleic acid comprises a polynucleotide encoding the VL of one of the antibodies provided herein. In another embodiment, the nucleic acid encodes the VH and VL of one of the antibodies provided herein. In certain embodiments, the nucleic acid molecule comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 30.

In a specific embodiment, the nucleic acid encodes a variant of one or more of the above-mentioned amino acid sequences (e.g., heavy chain and/or light chain amino acid sequences, or VH and/or VL amino acid sequences disclosed herein), wherein the variant has up to 25 amino acid modifications, such as up to 20, such as up to 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative substitutions.

Also provided are nucleic acid molecules having at least 80%, 85%, 90% (e.g., 95%, 96%, 97%, 98% or 99%) sequence identity to any of the foregoing sequences. Thus, for example, in certain embodiments, the nucleic acid comprises a nucleotide sequence encoding a heavy chain and/or light chain sequence or a VH and/or VL sequence of one of the antigen binding proteins disclosed herein.

Once the nucleic acids encoding the VH and VL segments are obtained, these nucleic acids can be further manipulated by standard recombinant DNA techniques, such as converting the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the nucleic acids encoding VL or VH can be operably linked to another nucleic acid encoding another polypeptide, such as an antibody constant region or a flexible linker.

The isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene by operably linking the nucleic acid encoding the VH to another nucleic acid molecule encoding the heavy chain constant region (hinge, CH1, CH2 and/or CH3). The sequence of the human heavy chain constant region gene is known in the art (see, e.g., Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and nucleic acid fragments comprising these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, e.g., an IgG1 region. For a Fab fragment heavy chain gene, the nucleic acid encoding the VH can be operably linked to another nucleic acid molecule encoding only the heavy chain CH1 constant region.

The isolated nucleic acid molecules encoding the VL region can be converted into full-length light chain genes (and Fab light chain genes) by operatively linking the nucleic acid molecules encoding the VL to another nucleic acid molecule encoding the light chain constant region CL. The sequence of human light chain constant region genes is known in the art (see, e.g., Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and nucleic acid fragments comprising these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

To generate an scFv gene, the nucleic acid fragments encoding VH and VL are operably linked to another fragment encoding a flexible linker (e.g., encoding the amino acid sequence (Gly ₄ -Ser) ₃ ) so that the VH and VL sequences can be expressed as a continuous single-chain protein in which the VL and VH regions are connected by a flexible linker (see, e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

In another aspect, nucleic acid molecules suitable for use as primers or hybridization probes for detecting nucleic acid sequences are also provided. The nucleic acid molecule may comprise only a portion of a nucleic acid sequence encoding a full-length polypeptide, for example, a fragment that can be used as a probe or primer or a fragment that encodes an active portion of a polypeptide (e.g., an ALPP and/or ALPPL2 binding portion).

Probes based on nucleic acid sequences can be used to detect nucleic acids or similar nucleic acids, such as transcripts encoding polypeptides. The probes can include labeling groups, such as radioisotopes, fluorescent compounds, enzymes, or enzyme cofactors. Such probes can be used to identify cells expressing polypeptides.

Also provided are vectors, including expression vectors, comprising one or more nucleic acids encoding one or more components of the ABP (e.g., VH and/or VL; and light and/or heavy chains). Expression vectors may include, but are not limited to, sequences that affect or control transcription, translation, and, if introns are present, RNA splicing of the coding region operably linked thereto. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, an optional operator sequence, a ribosome binding site, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

If necessary, the expression vector may further include a secretory signal peptide sequence operably linked to the target coding sequence so that the expressed polypeptide can be secreted from the recombinant host cells to more easily separate the target polypeptide from the cells.

Expression and cloning vectors of the present invention generally contain promoters that are recognized by the host organism and operably connected to the molecule encoding the polypeptide. A large number of promoters recognized by a variety of potential host cells are well known. The appropriate promoter is operably connected to the DNA encoding the heavy chain, light chain or other components of the antibody and antigen-binding fragment of the present invention by restriction enzyme digestion to remove the promoter and insert the desired promoter sequence into the vector. Suitable promoters for yeast hosts are also well known in the art. Yeast enhancers are advantageously used with yeast promoters. Suitable promoters for mammalian host cells are well known and include, but are not limited to, promoters obtained from viral genomes, such as polyoma viruses, fowl pox viruses, adenoviruses (such as adenovirus serotypes 2, 8 or 9), bovine papilloma viruses, avian sarcoma viruses, cytomegaloviruses, retroviruses, hepatitis B viruses and simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

Additional specific promoters that may be used include, but are not limited to, the SV40 early promoter (Benoist and Chambon, 1981, Nature 290:304-310); the CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad. U.S.A. 81:659-663); the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797); the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1444-1445); the promoter and regulatory sequences from the metallothionein gene (Prinster et al., 1982, Nature 290:304-310); the promoter from the SV40 early promoter (Benoist and Chambon, 1981, Nature 290:304-310); the promoter from the CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad. U.S.A. 81:659-663); the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797); the promoter from the SV40 early promoter (Benoist and Chambon, 1981, Nature 290:304-310 ... 296:39-42); and prokaryotic promoters such as the β-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25).

In certain embodiments, nucleic acids encoding different components of ABP can be inserted into the same expression vector. For example, nucleic acids encoding anti-ALPP/ALPPL2 antibody light chains or variable regions can be cloned into the same vector as nucleic acids encoding anti-ALPP/ALPPL2 antibody heavy chains or variable regions. In such embodiments, the two nucleic acids can be separated by an internal ribosome entry site (IRES) and under the control of a single promoter, the light chain and the heavy chain are expressed by the same mRNA transcript. Alternatively, the two nucleic acids can be under the control of two separate promoters, so that the light chain and the heavy chain are expressed by two separate mRNA transcripts. In some embodiments, nucleic acids encoding anti-ALPP/ALPPL2 antibody light chains or variable regions are cloned into one expression vector, and nucleic acids encoding anti-ALPP/ALPPL2 antibody heavy chains or variable regions are cloned into a second expression vector. In such embodiments, host cells can be co-transfected with two expression vectors to produce complete antibodies or antigen-binding fragments of the present invention.

B. Host Cells

After constructing the vector and inserting one or more nucleic acid molecules encoding the components of the ABP described herein into the appropriate site of the vector, the entire vector can be inserted into a suitable host cell for amplification and/or polypeptide expression.

Thus, in another aspect, a host cell comprising a nucleic acid molecule or vector such as described herein is also provided. In various embodiments, the ABP heavy chain and/or anti-light chain can be expressed in prokaryotic cells such as bacterial cells or eukaryotic cells such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. The selection of a suitable host cell depends on a variety of factors, such as the desired expression level, polypeptide modifications required or necessary for activity (such as glycosylation or phosphorylation), and the ease of folding into a biologically active molecule.

The introduction of one or more nucleic acids into the desired host cells can be accomplished by any method, including but not limited to calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described in, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected in the desired host cells according to any suitable method.

Exemplary prokaryotic host cells include eubacteria, such as Gram-negative or Gram-positive organisms, e.g., Enterobacteriaceae, such as Escherichia (e.g., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescans), and Shigella, as well as Bacillus (such as B. subtilis and B. licheniformis), Pseudomonas, and Streptomyces.

Yeast may also be used as a host cell, including but not limited to S. cerevisae, S. pombe, or Kluyveromyces lactis.

A variety of mammalian cell lines can be used as hosts, including, but not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese hamster ovary (CHO) cells, including CHOK1 cells (ATCC CRL61), DXB-11, DG-44, and Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216, 1980); SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59, 1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatoma cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TM cells (Mather et al., Annals N.Y Acad. Sci. 383: 44-68, 1982); MRC 5 cells or FS4 cells; mammalian myeloma cells and many other cell lines.

Once a suitable host cell is prepared, it can be used to express the desired ABP. Therefore, in another aspect, a method for producing the ABP as described herein is also provided. Generally, such methods include culturing a host cell comprising one or more expression vectors as described herein in a culture medium under conditions that allow expression of the ABP encoded by the one or more expression vectors; and recovering the ABP from the culture medium.

In some embodiments, the ABP is produced in a cell-free system. Non-limiting exemplary cell-free systems are described in, e.g., Sitaraman et al., Methods Mol. Biol. 498:229-44 (2009), Spirin, Trends Biotechnol. 22:538-45 (2004), Endo et al., Biotechnol. Adv. 21:695-713 (2003).

V. Antigen Binding Protein Conjugates

The ABP provided herein can be conjugated to a cytotoxic or cell growth inhibitory moiety (including its pharmaceutically compatible salt) to form a conjugate, such as an antibody drug conjugate (ADC). Particularly suitable moieties for conjugation with the ABP (e.g., antibody) are cytotoxic agents (e.g., chemotherapeutic agents), prodrug converting enzymes, radioisotopes or compounds, or toxins (these moieties are collectively referred to as therapeutic agents). For example, the ABP (e.g., anti-ALPP/ALPPL2 antibody) can be conjugated to a cytotoxic agent, such as a chemotherapeutic agent or a toxin (e.g., a cell growth inhibitor or a cytocidal agent, e.g., abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin). Examples of useful cytotoxic agent classes include, for example, DNA minor groove binders, DNA alkylating agents, and tubulin inhibitors. Exemplary cytotoxic agents include, for example, auristatins, camptothecins, calicheamicins, duocarmycins, etoposide, maytansines (e.g., DM1, DM2, DM3, DM4), taxanes, benzodiazepines Class (eg, pyrrolo[1,4]benzodiazepines Indole and benzodiazepines Oxazolidine benzodiazepines class) and vinca alkaloids.

In one embodiment, the ABP (e.g., anti-ALPP/ALPPL2 antibody) is conjugated to a prodrug converting enzyme. The prodrug converting enzyme can be recombinantly fused to the antibody or chemically conjugated thereto using known methods. Exemplary prodrug converting enzymes are carboxypeptidase G2, β-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase, nitroreductase, and carboxypeptidase A.

The technology of conjugating therapeutic agents to proteins, particularly antibodies, is well known. (See, e.g., Alley et al., Current Opinion in Chemical Biology 2010 14: 1-9; Senter, Cancer J., 2008, 14 (3): 154-169.) The therapeutic agent can be conjugated in a manner that reduces its activity unless it is cleaved from the antibody (e.g., by hydrolysis, by proteolytic degradation, or by a cleaving agent). In some aspects, the therapeutic agent is connected to the antibody via a cleavable linker that is sensitive to cleavage in the intracellular environment of a cancer cell expressing ALPP, but is substantially insensitive to the extracellular environment, such that when the conjugate is internalized by a cancer cell expressing ALPP (e.g., in an endosome, or, for example, by means of pH sensitivity or protease sensitivity, in a lysosomal environment or in a cell membrane caveolae environment), the conjugate is cleaved from the antibody. In some aspects, the therapeutic agent can also be connected to the antibody via a non-cleavable linker.

Typically, ADCs include a linker region between a therapeutic agent and an anti-ABP (e.g., an anti-ALPP/ALPPL2 antibody). The linker is typically cleavable under intracellular conditions, such that the cleavage of the linker releases the therapeutic agent from the antibody in the intracellular environment (e.g., in a lysosome or endosome or cell membrane caveolae). The linker can be, for example, a peptidyl linker cleaved by an intracellular peptidase or protease (including a lysosomal or endosomal protease). The cleavage agent can include cathepsins B and D and plasmin (see, e.g., Dubowchik and Walker, Pharm. Therapeutics 83: 67-123, 1999). The most typical is a peptidyl linker that can be cleaved by an enzyme present in a cell expressing ALPP. For example, a peptidyl linker (e.g., a linker comprising a Phe-Leu or Val-Cit peptide) that can be cleaved by a thiol-dependent protease cathepsin-B that is highly expressed in cancer tissue can be used.

Cleavable linkers can be pH sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used. (See, e.g., U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, Pharm. Therapeutics 83:67-123, 1999; Neville et al., Biol. Chem. 264:14653-14661, 1989.) Such linkers are relatively stable under neutral pH conditions (such as in blood), but are unstable below pH 5.5 or 5.0 (the approximate pH of lysosomes).

Other linkers are cleavable under reducing conditions (e.g., disulfide linkers). Disulfide linkers include those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio) butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyldithio) toluene), SPDB, and SMPT. (See, e.g., Thorpe et al., Cancer Res. 47:5924-5931, 1987; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel, ed., Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)

The linker can also be a malonate linker (Johnson et al., Anticancer Res. 15: 1387-93, 1995), a maleimidobenzoyl linker (Lau et al., Bioorg-Med-Chem. 3: 1299-1304, 1995), or a 3'-N-amide analog (Lau et al., Bioorg-Med-Chem. 3: 1305-12, 1995).

In other embodiments, the linker is a non-cleavable linker, such as a maleimido-alkylene- or maleimido-aryl linker, which is directly attached to the therapeutic agent and released by proteolytic degradation of the antibody.

Typically, the linker is substantially insensitive to the extracellular environment, meaning that when the ADC is present in an extracellular environment (e.g., in plasma), no more than about 20%, typically no more than about 15%, more typically no more than about 10%, even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linker in the ADC sample is cleaved. Whether the linker is substantially insensitive to the extracellular environment can be determined, for example, by incubating (a) the ADC ("ADC sample") and (b) an equimolar amount of an unconjugated antibody or therapeutic agent ("control sample") with plasma separately for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours), and then comparing the amount of the unconjugated antibody or therapeutic agent present in the ADC sample to the amount present in the control sample, as measured, for example, by high performance liquid chromatography.

The linker can also promote cellular internalization. When conjugated to a therapeutic agent (i.e., in the context of a linker-therapeutic agent portion of an ADC or ADC derivative as described herein), the linker can promote cellular internalization. Alternatively, when conjugated to both a therapeutic agent and an antigen binding protein (e.g., an anti-ALPP/ALPPL2 antibody) (i.e., in the context of an ADC as described herein), the linker can promote cellular internalization.

Exemplary antibody-drug conjugates include antibody-drug conjugates based on auristatins, which means that the drug component is an auristatin drug. Auristatins bind to tubulin, have been shown to interfere with microtubule dynamics and nuclear and cell division, and have anti-cancer activity. Typically, an antibody-drug conjugate based on auristatins includes a linker between an auristatin drug and an ABP (e.g., an anti-ALPP/ALPPL2 antibody). The linker can be, for example, a cleavable linker (e.g., a peptidyl linker) or a non-cleavable linker (e.g., a linker released by antibody degradation). Auristatin can be auristatin E or a derivative thereof. Auristatin can be, for example, an ester formed between auristatin E and a ketoacid. For example, auristatin E can react with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include MMAF and MMAE. The synthesis and structure of exemplary auristatins are described in U.S. Pub. Nos. 7,659,241, 7,498,298, 2009-0111756, 2009-0018086, and 7,968,687, each of which is incorporated herein by reference in its entirety and for all purposes.

Exemplary auristatin-based antibody drug conjugates include mc-vc-PABC-MMAE (also referred to herein as vcMMAE or 1006), mc-vc-PABC-MMAF, mc-MMAF, and mp-dLAE-PABC-MMAE (also referred to herein as dLAE-MMAE or mp-dLAE-MMAE or 7092), the antibody drug conjugates being shown below, wherein Ab is an ABP (e.g., an anti-ALPP/ALPPL2 antibody as described herein) and val-cit(vc) represents a valine-citrulline dipeptide, and dLAE represents a D-leucine-alanine-glutamic acid tripeptide:

Tripeptide:

Or a pharmaceutically acceptable salt thereof. The drug loading is represented by p, i.e., the number of drug-linker moieties per antibody. Depending on the context, p can represent the average number of drug-linker moieties per antibody in the antibody composition, also referred to as the average drug loading. The range of p is 1 to 20 and preferably 1 to 12 or 1 to 8. In some preferred embodiments, when p represents the average drug loading, the range of p is about 2 to about 5. In some embodiments, p is about 2, about 3, about 4 or about 5. The average number of drugs per antibody in the formulation can be characterized by conventional means such as mass spectrometry, HIC, ELISA assays and HPLC. In some aspects, the ABP (e.g., anti-ALPP/ALPPL2 antibody) is connected to the drug-linker via a cysteine residue of the antibody. In some embodiments, the cysteine residue is a cysteine residue engineered into the antibody. In other aspects, the cysteine residue is an interchain disulfide bond cysteine residue.

VI. Therapeutic Applications

A. Methods of treating diseases

On the other hand, methods for treating disorders (e.g., cancer) associated with cells expressing ALPP and/or ALPPL2 are provided. These cells may or may not express elevated levels of ALPP and/or ALPPL2 relative to cells not associated with the disorder of interest. Therefore, certain embodiments relate to using the ABP described herein (e.g., anti-ALPP/ALPPL2 antibodies) as naked antibodies or as conjugates (e.g., antibody drug conjugates) to treat subjects, such as subjects with cancer. In some of these embodiments, the method includes administering an effective amount of an ABP (e.g., anti-ALPP/ALPPL2 antibody) or conjugate (e.g., anti-ALPP/ALPPL2 ADC) or a composition comprising such an ABP or conjugate to a subject in need. In certain exemplary embodiments, the method includes treating cancer in cells, tissues, organs, animals, or patients. Most typically, the method of treatment includes treating human cancer. In some embodiments, treatment involves monotherapy. In other methods, the antigen binding protein is administered as part of a combination therapy with one or more other therapeutic agents, surgery, and/or radiation.

The positive treatment effect of cancer can be measured in a variety of ways (see, e.g., W.A.Weber, J.Null.Med.50:1S-10S (2009); and Eisenhauer et al., Eur.J Cancer45:228-247 (2009)). In some embodiments, the response to treatment with ABP or conjugate is assessed using the RECIST 1.1 criteria. In some embodiments, the treatment achieved by a therapeutically effective amount is any one of inhibition of further tumor growth, induction of tumor regression, partial response (PR), complete response (CR), progression-free survival (PFS), disease-free survival (DFS), objective response (OR), or overall survival (OS). In some embodiments, treatment delays or prevents the occurrence of metastasis. The progress of treatment can be monitored using a variety of methods. For example, inhibition can result in a decrease in tumor size and/or a decrease in metabolic activity within the tumor. Both parameters can be measured, for example, by MRI or PET scans. Inhibition can also be monitored by biopsy to determine the level of necrosis, tumor cell death, and intratumoral vascularity. Dosage regimens for the therapies described herein that are effective in treating cancer patients may vary depending on factors such as the patient's disease state, age, and weight, and the ability of the therapy to elicit an anti-cancer response in the subject. Although embodiments of the methods of treatment, medicaments, and uses of the invention may not be effective in achieving a positive therapeutic effect in every subject, it should do so in a statistically significant number of subjects, as determined by any statistical test known in the art, such as Student's t-test, chi2 test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

As used herein, "RECIST 1.1 response criteria" refers to the definitions set forth in Eisenhauer et al., Eur. J Cancer 45:228-247 (2009) for target lesions or non-target lesions, as appropriate, in the context of measuring response.

An effective amount of an ABP (e.g., anti-ALPP/ALPPL2 antibody) or ADC can be administered in one or more administrations, applications or dosages, and is not intended to be limited to a particular formulation or route of administration. Typically, the therapeutically effective amount of the active ingredient is in the range of 0.1 mg/kg to 100 mg/kg, e.g., 1 mg/kg to 100 mg/kg, 1 mg/kg to 10 mg/kg.

Exemplary dosages of an ABP (e.g., an anti-ALPP/ALPPL2 antibody) are, for example, 0.1 mg/kg to 50 mg/kg of patient body weight, more typically 1 mg/kg to 30 mg/kg, 1 mg/kg to 20 mg/kg, 1 mg/kg to 15 mg/kg, 1 mg/kg to 12 mg/kg, or 1 mg/kg to 10 mg/kg, or 2 mg/kg to 30 mg/kg, 2 mg/kg to 20 mg/kg, 2 mg/kg to 15 mg/kg, 2 mg/kg to 12 mg/kg, or 2 mg/kg to 10 mg/kg, or 3 mg/kg to 30 mg/kg, 3 mg/kg to 20 mg/kg, 3 mg/kg to 15 mg/kg, 3 mg/kg to 12 mg/kg, or 3 mg/kg to 10 mg/kg.

Exemplary dosages of ABP (e.g., anti-ALPP/ALPPL2 antibodies) are, for example, 0.01 mg/kg to 10 mg/kg, 0.1 mg/kg to 10 mg/kg, 0.3 mg/kg to 3 mg/kg, 0.5 mg/kg to 3 mg/kg, 1 mg/kg to 7.5 mg/kg, or 2 mg/kg to 7.5 mg/kg, or 3 mg/kg to 7.5 mg/kg of subject body weight, or 0.1-20 or 0.5-5 mg/kg body weight (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg), or 10-1500 or 200-1500 mg as a fixed dose. In some methods, a dose of at least 1.5 mg/kg, at least 2 mg/kg, or at least 3 mg/kg is administered to the patient once every three weeks or longer.

The dose administered can vary according to known factors, such as the pharmacodynamic characteristics of a particular agent and its mode and route of administration; the age, health status and weight of the recipient; the type and extent of the disease or indication to be treated, the nature and extent of the symptoms, the type of concurrent treatment, the frequency of treatment and the desired effect. The initial dose can be increased to exceed the upper limit level to quickly reach the desired blood level or tissue level. Alternatively, the initial dose can be less than the optimal value, and the daily dose can be gradually increased during the treatment.

The frequency of administration depends on factors such as the half-life of the ABP or ADC in the circulation, the patient's condition and the route of administration. The frequency can be, for example, daily, weekly, monthly, quarterly or in response to changes in the patient's condition or the progress of the cancer being treated at irregular intervals. An exemplary frequency of intravenous administration is between twice a week and once a quarter during continuous treatment, but higher or lower frequency administration is also possible. Other exemplary frequencies of intravenous administration are weekly, every other week, three weeks or every three weeks in every four weeks during continuous treatment, but higher or lower frequency administration is also possible. For subcutaneous administration, an exemplary administration frequency is daily to monthly, but higher or lower frequency administration is also possible.

The number of doses administered depends on the nature of the cancer (e.g., whether acute or chronic symptoms are present) and the reaction of the disease to treatment. In some aspects, for acute conditions or acute exacerbations of chronic conditions, 1 to 10 doses are generally sufficient. Sometimes, a single bolus dose optionally in a separate form is sufficient for acute conditions or acute exacerbations of chronic conditions. For recurrence or acute exacerbations of acute conditions, treatment can be repeated. For chronic conditions, antibodies can be administered at regular intervals, such as weekly, biweekly, monthly, quarterly, every six months, for at least 1, 5 or 10 years, or the patient's lifetime.

Exemplary cancers suitable for treatment with the antigen binding proteins provided herein are those with ALPP and/or ALPPL2 expression in cancer cells or tissues. Examples of cancers that can be treated with ABP or its conjugates include, but are not limited to, hematopoietic tumors, hematopoietic tumors that give rise to solid tumors, solid tumors, soft tissue tumors, and metastatic lesions.

Exemplary solid tumors that can be treated include, but are not limited to, malignancies of multiple organ systems, such as adenocarcinomas and carcinomas, such as those affecting the head and neck (including pharynx), lung (small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC)), breast, gastrointestinal tract (e.g., oral cavity, esophagus, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), genital and urogenital tract (e.g., kidney, urothelium, bladder, ovary, uterus, cervix, endometrium, prostate, testis), skin (e.g., melanoma), etc. In certain embodiments, the solid tumor is an NMDA receptor positive teratoma. In other embodiments, the cancer is selected from breast cancer, colon cancer, pancreatic cancer (e.g., pancreatic neuroendocrine tumor (PNET) or pancreatic ductal adenocarcinoma (PDAC)), gastric cancer, uterine cancer, and ovarian cancer. In some embodiments, the cancer is a malignant testicular germ cell tumor (GCT) or a malignant ovarian GCT. In other embodiments, the cancer is not a pure teratoma. In some embodiments, the solid tumor cancer is metastatic. In some embodiments, the sliding tumor cancer cannot be removed by surgery (unresectable).

In certain embodiments, cancer is a solid tumor associated with ascites. Ascites is a symptom of many types of cancers, and may also be caused by a variety of illnesses, such as advanced liver disease. The types of cancers that may cause ascites include but are not limited to breast cancer, lung cancer, colorectal cancer (colon cancer), gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer (endometrial cancer), peritoneal cancer, etc. In some embodiments, the solid tumor associated with ascites is selected from breast cancer, colon cancer, pancreatic cancer, gastric cancer, uterine cancer and ovarian cancer. In some embodiments, cancer is associated with pleural effusion, such as lung cancer.

In specific embodiments, the cancer is HGSOC, wherein the HGSOC progresses or relapses in the patient within six months after prior platinum-containing chemotherapy, and the patient has received one to three lines of prior anticancer therapy, including at least one line of therapy containing bevacizumab or a bevacizumab biosimilar. In other embodiments, the cancer is NSCLC, wherein the patient has unresectable locally advanced or metastatic NSCLC and has received platinum-based therapy and a PD-L1 inhibitor. In other embodiments, the cancer is gastric cancer, wherein the patient has unresectable locally advanced or metastatic gastric cancer and has previously received platinum- and fluoropyrimidine-based chemotherapy.

In specific embodiments, the cancer is ovarian cancer, lung cancer, endometrial cancer, bladder cancer, or gastric cancer.

B. Combination Therapy

The methods, antigen binding proteins and conjugates described herein can be used in combination with other therapeutic agents and/or methods. In such a combined treatment method, two (or more) different treatments are delivered to the subject during the process of the subject being afflicted with the disease, so that the effect of the treatment on the patient overlaps at a certain time point. In certain embodiments, the delivery of one treatment still occurs when the delivery of the second treatment begins, so that there is overlap in terms of administration. This is sometimes referred to as "simultaneous" or "parallel delivery" in this article. In other embodiments, the delivery of one treatment ends before the delivery of another treatment begins. In some embodiments of any one case, due to combined administration, the treatment is more effective. For example, the second treatment is more effective, for example, compared with the application of the second treatment in the absence of the first treatment, an equivalent effect is observed with less second treatment, or the second treatment alleviates symptoms to a greater extent, or a similar situation is observed with the first treatment. In some embodiments, delivery causes the alleviation of symptoms or other parameters associated with the disease to be greater than the alleviation observed when delivering one treatment in the absence of another treatment. The effects of the two treatments can be partially added, completely added, or greater than added (i.e., synergistic reaction). Delivery can make it possible to still detect the effect of the first treatment delivered when the second treatment is delivered.

In certain embodiments, the methods provided herein include administering to a subject an ABP (e.g., an anti-ALPP/ALPPL2 antibody) or ADC described herein, such as a composition or formulation, in combination with one or more additional therapies such as surgery, radiotherapy, or administration of another therapeutic formulation. For example, in some embodiments, the ABP is combined with chemotherapy (e.g., a cytotoxic agent), targeted therapy (e.g., an antibody to a cancer antigen), an angiogenesis inhibitor, and/or an immunomodulator (such as an inhibitor of an immune checkpoint molecule). In other embodiments, the additional therapy is an anti-inflammatory agent (e.g., methotrexate) or an anti-fibrotic agent. ABP (e.g., an anti-ALPP/ALPPL2 antibody) or ADC and additional therapies can be administered simultaneously or sequentially.

In some embodiments, exemplary cytotoxic agents that can be used in combination with the ABP include anti-microtubule agents, topoisomerase inhibitors, antimetabolites, protein synthesis and degradation inhibitors, mitotic inhibitors, alkylating agents, platinating agents, nucleic acid synthesis inhibitors, histone deacetylase inhibitors (HDAC inhibitors, e.g., vorinostat (SAHA, MK0683), entinostat (MS-275), panobinostat (LBH589), trichostatin A (TSA), mosinostat (MGCD0103), belinostat (PXD101), romidepsin (FK228, depsipeptide)), DNA methyltransferase inhibitors, nitrogen mustards, nitrosoureas, ethyleneimines, alkyl sulfonates, triazenes, folic acid analogs, nucleoside analogs, ribonucleotide reductase inhibitors, vinca alkaloids, taxanes, epothilones, intercalating agents, agents capable of interfering with signal transduction pathways, agents that promote apoptosis and radiation, or antibody molecule conjugates that bind to surface proteins to deliver toxic agents. In one embodiment, the cytotoxic agent that can be administered with the ABP described herein is a platinum-based agent (such as cisplatin), cyclophosphamide, dacarbazine, methotrexate, fluorouracil, gemcitabine, capecitabine, hydroxyurea, topotecan, irinotecan, azacytidine, vorinostat, ixabepilone, bortezomib, taxanes (e.g., paclitaxel or docetaxel), cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, vinorelbine, colchicine, anthracycline (e.g., doxorubicin or epirubicin), daunorubicin, dihydroxyanthraxandione, mitoxantrone, mithramycin, actinomycin D, doxorubicin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, ricin, or a maytansine.

In some embodiments, the antigen binding protein is administered as part of a chemotherapy regimen, such as CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone); CVP (cyclophosphamide, vincristine, and prednisone); RCVP (rituximab + CVP); RCHOP (rituximab + CHOP); RCHP (rituximab, cyclophosphamide, doxorubicin, and prednisone); RICE (rituximab + ifosfamide, carboplatin, etoposide); RDHAP (rituximab + dexamethasone, cytarabine, cisplatin); RESHAP (rituximab + etoposide, methylprednisolone, cytarabine, cisplatin); R-BENDA (rituximab and bendamustine), RGDP (rituximab, gemcitabine, dexamethasone, cisplatin). In one embodiment, one of CHOP, CVP, RCVP, RCHOP, RCHP, RICE, RDHAP, RESHAP, R-BENDA, and RGDP is administered in combination therapy with an antigen binding protein or conjugate as described herein.

In certain embodiments, examples of targeted therapies that can be combined with ABPs include, but are not limited to, the use of therapeutic antibodies. Exemplary antibodies include, but are not limited to, those that bind to cell surface proteins such as Her2, CDC20, CDC33, mucin-like glycoproteins, and epidermal growth factor receptor (EGFR) present on tumor cells and optionally induce cell growth inhibition and/or cytotoxic effects on tumor cells displaying these proteins. Exemplary antibodies also include (trastuzumab), which is used to treat breast cancer and other forms of cancer, and (rituximab), (ibritumomab tiuxetan), and (epatuzumab), which can be used to treat non-Hodgkin lymphoma and other forms of cancer. Other exemplary antibodies include panitumumab (IMC-C225);ertinolib(Iressa); (iodine-131 tositumomab); KDR (kinase domain receptor) inhibitors; anti-VEGF antibodies and antagonists (e.g., Antibodies to VEGF receptors and antigen binding regions; Antibodies to Ang-1 and Ang-2 and antigen binding regions; Antibodies to Tie-2 and other Ang-1 and Ang-2 receptors; Tie-2 ligands; Antibodies to Tie-2 kinase inhibitors; Hif-1a and In some embodiments, the cancer therapeutic agent is a polypeptide that selectively induces apoptosis of tumor cells, including but not limited to the TNF-related polypeptide TRAIL.

In certain embodiments, the antigen binding proteins provided herein are used in combination with one or more anti-angiogenic agents that reduce angiogenesis. Such agents include, but are not limited to, IL-8 antagonists; B-FGF; FGF antagonists; Tek antagonists (Cerretti et al., U.S. Publication No. 2003/0162712; Cerretti et al., U.S. Patent No. 6,413,932; and Cerretti et al., U.S. Patent No. 6,521,424); anti-TWEAK agents (including, but not limited to, antibodies and antigen binding regions); soluble TWEAK receptor antagonists (Wiley, U.S. Patent No. 6,727,225); ADAM disintegrin domains for antagonizing integrins binding of an ephrin to its ligand (Fanslow et al., U.S. Publication No. 2002/0042368); anti-eph receptor and anti-ephrin antibodies, antigen binding regions or antagonists (U.S. Pat. Nos. 5,981,245; 5,728,813; 5,969,110; 6,596,852; 6,232,447; 6,057,124); anti-VEGF agents (e.g., antibodies or antigen binding regions that specifically bind to VEGF, or soluble VEGF receptors or their ligand binding regions), such as or VEGF-TRAP ^™ ; and anti-VEGF receptor agents (eg, antibodies or antigen-binding regions that specifically bind thereto); EGFR inhibitors (eg, antibodies or antigen-binding regions that specifically bind thereto), such as panitumumab, (gefitinib), (erlotinib); anti-Ang-1 and anti-Ang-2 agents (e.g., antibodies or antigen-binding regions that specifically bind thereto or their receptors, e.g., Tie-2/TEK); and anti-Tie-2 kinase inhibitors (e.g., antibodies or antigen-binding regions that specifically bind to and inhibit the activity of growth factors, such as antagonists of hepatocyte growth factor (HGF, also known as scatter factor), and antibodies or antigen-binding regions that specifically bind to its receptor "c-met"; anti-PDGF-BB antagonists; antibodies and antigen-binding regions of PDGF-BB ligands; and PDGFR kinase inhibitors.

Other anti-angiogenic agents that can be used in combination with the antigen binding proteins include agents such as MMP-2 (matrix metalloproteinase 2) inhibitors, MMP-9 (matrix metalloproteinase 9) inhibitors, and COX-II (cyclooxygenase II) inhibitors. Examples of useful COX-II inhibitors include (celecoxib), valdecoxib, and rofecoxib.

As used herein, "immune checkpoint molecules" refer to molecules in the immune system that upregulate signals (stimulatory molecules) and/or downregulate signals (inhibitory molecules). Many cancers evade the immune system by inhibiting T cell signaling. In certain embodiments, exemplary immune checkpoint molecules that can be used with ABP include, but are not limited to, programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), PD-L2, cytotoxic T lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1), CEACAM-5, V domain Ig inhibitor of T cell activation (VISTA), B and T lymphocyte attenuation factor (BTLA), T cell immune receptor with Ig and ITIM domains (TIG IT), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), CD160, TGFR, adenosine 2A receptor (A2AR), B7-H3 (also known as CD276), B7-H4 (also known as VTCN1), indoleamine 2,3-dioxygenase (IDO), 2B4, killer cell immunoglobulin-like receptor (KIR), OX40, 4-1BB, 4-1BBL, B7-H3, inducible T-cell co-stimulator (ICOS/ICOS-L), CD27/CD70, glucocorticoid-induced TNF receptor (GITR), CD47/signal regulatory protein α (SIRPα), and indoleamine-2,3-dioxygenase (IDO).

In certain embodiments, specific examples of immune checkpoint inhibitors that can be used in combination with ABPs include, but are not limited to, the following monoclonal antibodies: PD-1 inhibitors such as pembrolizumab ( Merck) and nivolumab ( Bristol-Myers Squibb; PD-L1 inhibitors such as atezolizumab ( Genentech), Avelumab ( Pfizer), durvalumab ( AstraZeneca); and CTLA-1 inhibitors such as ipilimumab ( Bristol-Myers Squibb) and tremelimumab (AstraZeneca).

VII. Diagnostic Applications

In another aspect, the ABPs (eg, anti-ALPP/ALPPL2 antibodies or fragments thereof), polypeptides and nucleic acids provided herein can be used in methods for detecting, diagnosing and monitoring diseases, disorders or conditions associated with ALPP and/or ALPPL2.

In some embodiments, the method comprises detecting the expression of ALPP and/or ALPPL2 in a sample obtained from a subject suspected of having a condition associated with ALPP and/or ALPPL2. In some embodiments, the detection method comprises contacting the sample with an antibody, polypeptide, or polynucleotide as described herein and determining whether the level of binding is different from the level of binding of a reference or comparison sample. In some embodiments, such methods can be used to determine whether an antibody or polypeptide described herein is an appropriate treatment for a subject.

For example, in one embodiment, a cell or cell/tissue lysate is contacted with an anti-ALPP/ALPPL2 antibody and binding between the antibody and the cell or antigen is determined. When the test cell shows binding activity compared to a reference cell of the same tissue type, it can indicate the presence of a disease or condition associated with ALPP and/or ALPPL2. In some embodiments, the test cell is from human tissue.

Various methods known in the art for detecting specific antibody-antigen binding can be used. Exemplary immunoassays that can be performed according to the present invention include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), turbidity inhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).

The diagnostic applications provided herein include the use of ABPs (e.g., anti-ALPP/ALPPL2 antibodies or fragments thereof) to detect the expression of ALPP and/or ALPPL2 and the binding of ligands to ALPP and/or ALPPL2. For diagnostic applications, the ABP is typically labeled with a detectable labeling group. Suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g., ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I), fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzyme groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotin groups, or predetermined polypeptide epitopes recognized by a second reporter gene (e.g., leucine zipper pair sequences, binding sites of secondary antibodies, metal binding domains, epitope tags). In some embodiments, the labeling group is coupled to the ABP via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used.Examples of methods that can be used to detect the presence of ALPP and/or ALPPL2 include, for example, the immunoassays described above.

In another aspect, the ABP can be used to identify one or more cells expressing ALPP and/or ALPPL2. In a specific embodiment, the antigen binding protein is labeled with a labeling group, and the binding of the labeled antigen binding protein to ALPP and/or ALPPL2 is detected. In another specific embodiment, the binding of the antigen binding protein to ALPP and/or ALPPL2 is detected in vivo.

Antigen binding proteins (eg, anti-ALPP/ALPPL2 antibodies or fragments thereof) can also be used as staining reagents in pathology using techniques well known in the art.

VIII. Pharmaceutical Compositions and Formulations

Pharmaceutical compositions comprising an ABP (e.g., an anti-ALPP/ALPPL2 antibody or fragment thereof) are also provided and can be used in any therapeutic application disclosed herein. In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of one or more antigen binding proteins, and a pharmaceutically acceptable diluent or carrier. In other embodiments, the pharmaceutical composition comprises a therapeutically effective amount of one or more antigen binding proteins, a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant. Acceptable formulation materials are non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition can be formulated as a liquid, frozen or lyophilized composition.

In certain embodiments, the pharmaceutical composition may contain formulation materials for changing, maintaining or preserving, for example, pH, osmotic pressure, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids; antimicrobial agents; antioxidants; buffers; bulking agents; chelating agents; complexing agents; fillers; carbohydrates such as monosaccharides or disaccharides; proteins; colorants, flavoring agents and diluents; emulsifiers; hydrophilic polymers; low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives; solvents (such as glycerol, propylene glycol or polyethylene glycol); sugar alcohols; suspending agents; surfactants or wetting agents; stability enhancers; tension enhancers; delivery vehicles; and/or pharmaceutical adjuvants. Further details and selection of suitable agents that can be incorporated into pharmaceutical compositions are provided, for example, in Remington's Pharmaceutical Sciences, 22nd edition, (Loyd V. Allen, ed.) Pharmaceutical Press (2013), Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th edition, Lippencott Williams and Wilkins (2004), and Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd edition, Pharmaceutical Press (2000).

The selection of the components of the pharmaceutical composition depends on, for example, the intended route of administration, delivery form, and desired dosage. See, for example, Remington's Pharmaceutical Sciences, 22nd edition, (Loyd V.Allen, ed.) Pharmaceutical Press (2013). Select compositions to affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the disclosed antigen-binding proteins. The main vehicle or carrier in the pharmaceutical composition can be aqueous or non-aqueous. For example, a suitable vehicle or carrier can be water for injection or a physiological saline solution. In certain embodiments, the antigen-binding protein composition can be prepared for storage by mixing the selected composition with the desired purity with an optional formulation agent in the form of a lyophilized cake or an aqueous solution. In addition, in certain embodiments, the antigen-binding protein can be formulated as a lyophilized product using a suitable excipient.

The pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal and rectal administration. The preferred route of administration for antigen binding proteins (e.g., antibodies) is IV infusion. In another preferred embodiment, the preparation is administered by intramuscular or subcutaneous injection.

IX. Kits/Products

Also provided are kits containing the ABP described herein. In one embodiment, such kits comprise one or more containers comprising an antigen binding protein (e.g., an anti-ALPP/ALPPL2 antibody) or a unit dosage form and/or preparation. In some embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising an antigen binding protein, with or without one or more additional agents. In some embodiments, such a unit dose is provided in a disposable prefilled syringe for injection. In various embodiments, the composition contained in the unit dose may comprise: saline; a buffer, other formulation components, and/or be formulated within a stable and effective pH range as described herein. Alternatively, in some embodiments, the composition is provided in the form of a lyophilized powder, which can be reconstituted after adding an appropriate liquid (e.g., sterile water).

Some kits as provided herein also contain instructions for treating diseases associated with ALPP and/or ALPPL2 (such as ovarian cancer) according to any of the methods described herein. The kit may also contain instructions for how to select or identify individuals suitable for treatment. The instructions provided in the kits of the present invention are typically written instructions on a label or package insert (e.g., a sheet of paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. In some embodiments, the kit further comprises another therapeutic agent, such as the above-mentioned therapeutic agent suitable for use in combination with the antigen binding protein.

In another aspect, a kit for detecting the presence of ALPP and/or ALPPL2 or cells expressing ALPP and/or ALPPL2 in a sample is provided. Such kits generally comprise an antigen binding protein as described herein and instructions for use of the kit.

Certain kits are used, for example, for diagnosing cancer and include containers containing antigen binding proteins (e.g., anti-ALPP/ALPPL2 antibodies) and one or more reagents for detecting binding of the antigen binding protein to ALPP and/or ALPPL2. Such reagents may include, for example, fluorescent labels, enzyme labels, or other detectable labels. Reagents may also include secondary or tertiary antibodies or reagents, such as enzyme reactions for producing products that can be visualized. In one embodiment, the diagnostic kit includes one or more antigen binding proteins in a labeled or unlabeled form in a suitable container, an incubation reagent for indirect assay, and a substrate or derivatizing agent for detection in such an assay, depending on the nature of the label.

Kits such as those provided herein can be used for in situ detection. Some methods utilizing such kits include removing a histological specimen from a patient and then combining a labeled antigen binding protein (e.g., an anti-ALPP/ALPPL2 antibody) with the biological sample. Using such methods, not only can the presence of ALPP or ALPP fragments and/or ALPPL2 or ALPPL2 fragments be determined, but also the distribution of such peptides in the tissue being examined (e.g., in the case of assessing the spread of cancer cells) can be determined.

In another aspect, an anti-idiotypic antibody (Id) that binds to an antigen binding protein (e.g., an anti-ALPP/ALPPL2 antibody) is provided. The Id antibody can be prepared by immunizing an animal of the same species and genotype as the source of the anti-ALPP/ALPPL2 mAb with the mAb used to prepare the anti-Id. The immunized animal can generally recognize and react to the idiotypic determinants of the immunized antibody by producing antibodies (anti-Id antibodies) directed against these idiotypic determinants.

The following examples, including the experiments performed and results obtained, are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims.

X. Examples

Example 1: ALPP/ALPPL2 expression levels

The cell lines described in the following examples are maintained in culture according to the American Type Culture Collection (ATCC) or the German Collection of Microorganisms and Cell Cultures (DMSZ), the Japan Cancer Research Resource Bank (JCRB), or other known conditions.

The ALPP copy number on the cell surface of various cancer cell lines was quantified using mouse ALPP mAb as primary antibody and DAKO QiFiKit flow cytometry indirect assay (as described by the manufacturer (DAKO A/S, Glostrup, Denmark) and evaluated with Attune NxT flow cytometer. The resulting number of ALPP molecules expressed per cell is shown in Table 3. ALPP/ALPPL2 mRNA expression levels were obtained from Genentech cell line RNA-seq data (see Klijn C et al. Nat Biotechnol. 2015 Mar; 33(3): 306-12).

Table 3: ALPP/ALPPL2 molecules/cells in various cell lines

Tumor tissue arrays were obtained from commercial sources. Frozen or formalin-fixed and paraffin-embedded (FFPE) tissues were purchased from USBiomax Inc. All samples were processed on a Bond-Max ^™ automated stainer (Leica).

The frozen samples sliced on the slides were fixed with acetone for 10 minutes before staining. The slides were incubated with anti-ALPP/ALPPL2 primary antibodies (H17E2; Thermo; Catalog No. MA1-20245). Isotype-matched mouse IgG1 was used as a negative control for background staining. For automatic IHC staining, we used the Refine DAB kit (Leica, catalog No. DS9800). The slides were incubated with 5 μg/ml of mouse monoclonal primary antibodies against ALPP for 45 minutes, and pre-incubated with PeroxAbolish (Biocare Medical catalog No. PXA969M) reagent for 15 minutes, and then pre-incubated with protein blocks (DAKO catalog No. X0909) for 20 minutes. The FFPE slides sliced on the slides were dewaxed and rehydrated at 72°C using Bond ^TM Dewax solution (Leica, catalog No. AR9222). Antigen retrieval was performed at 95°C-100°C for 20 minutes using EDTA-based Bond ^TM epitope retrieval solution 2 (Leica, catalog number AR9640), followed by incubation with anti-ALPP/ALPPL2 primary antibody (25C3 monoclonal Ab; mouse monoclonal developed in-house) at 1 μg/ml for 45 minutes. Isotype-matched mouse IgG2a was used as a negative control for background staining. For automated IHC staining, we used the Refine DAB kit (Leica, catalog number DS9800). Slides were incubated with 1 μg/ml of mouse monoclonal antibody against ALPP mAb for 20 minutes and then incubated with protein block (DAKO catalog number X0909) for 45 minutes. After color development, the sections were counterstained with hematoxylin and coverslipped. Slides were evaluated and scored by a pathologist, and images were taken using an Aperio slide scanner (Leica). Staining intensity was scored from 0 to +3, and frequencies were expressed in quartiles (0-25, 26-50, 51-75, and 76-100). ALPP/ALPPL2 expression was found to be high in multiple solid tumor indications including ovarian, testicular, and endometrial, as shown in Table 4. ALPP/ALPPL2 expression was also present in ~25% of lung adenocarcinoma, gastric cancer, and bladder cancer samples.

Table 4

9

Example 2: Lead Antibody Selection

Conjugation and in vitro cytotoxicity

Mice were immunized with recombinant full-length ALPPL2. Lymphocytes collected from the spleen and lymph nodes of mice producing ALPP antibodies were fused with myeloma cells. The fused cells were recovered overnight in hybridoma growth medium. After recovery, the cells were centrifuged and then plated in semi-solid medium. The hybridomas were incubated and hybridoma clones producing IgG were selected. Antibodies from this hybridoma series were screened on HEK293 cell lines expressing ALPP, ALPPL2, ALPI and ALPL using iQue according to the manufacturer's instructions. Antibodies with cross-reactivity to ALPP and ALPPL2 (but not ALPI and ALPL) were evaluated as ADCs.

Various mouse anti-ALPP/ALPPL2 monoclonal mouse antibodies were conjugated with 10-12 loaded MDpr-PEG(12)-gluc-MMAE or Auristatin T, which exhibited bystander activity or did not exhibit bystander activity, respectively. The conjugation method is described in U.S. Publication No. 2018/0092984.

CAOV3 (ALPP), COV644 (ALPP+) and NCI-H1651 (ALPPL2++ALPP+) tumor cells were incubated with ALPP/ALPPL2 antibody drug conjugates (ADC) at 37°C for 96 hours. Human IgG ADC was used as a negative control. Cell viability was measured using Cell Titer Glo according to the manufacturer's instructions. Fluorescence signals were measured on a Fusion HT fluorescent microplate reader (PerkinElmer, Waltham, MA). The data were normalized to untreated cells and the x50 values were calculated using Graph Pad software. As shown in Figures 1 to 2, the Ab subgroups showed lower x50 values, with two payloads indicating higher drug delivery capabilities.

Flow cytometry and saturation binding assay

Specificity and binding affinity were evaluated with HEK293 cells expressing cynomolgus ALPP, human ALPP, ALPPL2, ALPI and ALPL. In brief, one hundred thousand target-expressing HEK293 cells were transferred to 96-well plates. Cells were precipitated by centrifugation and resuspended in 100 μL PBS + 2% w / v BSA. After blocking, the cells were resuspended in PBS + 2% w / v BSA, where the concentration of unlabeled monoclonal anti-ALPP / ALPPL2 antibodies ranged from 8pM to 666nM and incubated on ice for 30 minutes. The cells were washed twice in PBS and resuspended in R-PE-labeled goat anti-human or anti-mouse secondary antibodies (Jackson Immunoresearch, West Grove, PA) on ice for 30 minutes. The specificity of the monoclonal antibodies to human and cynomolgus ALPP and ALPPL2 was confirmed by flow cytometry, but there was no specificity for other members of the alkaline phosphatase family. Fluorescence was analyzed using an Attune NxT flow cytometer, and the percentage of saturated fluorescent signal was used to determine the percent binding and subsequently calculate the apparent _KD . Antibodies 1C7 and 12F3 showed the lowest _KD among the top candidates, as shown in Figure 3. However, while SG82-12F3 and SG84-1F7 showed similar affinities to their targets, SG82-12F3 showed higher saturation levels for ALPP than SG84-1F7, as shown in Figure 4. Ultimately, the 12F3 antibody was selected for humanization based on its superior ADC cytotoxicity and high affinity for ALPP/ALPPL2 and having an epitope that is conserved with the cynomolgus monkey ortholog.

Example 3: Humanization and binding studies

Humanized antibodies are derived from the mouse 12F3 antibody. Eight humanized heavy chains (HA-HH) and twelve humanized light chains (L1-LI) with back mutations incorporated at different positions were prepared. In some cases, the back mutations matched the mouse germline, but in other cases they did not (such as the case of somatic mutations). The humanized heavy and light chains were paired. See the sequence alignments of Figures 5 to 8 and the specific mutations of Tables 5 to 8.

Table 5: Humanizing mutations in h12F3 variable heavy (vH) chain variants

Table 6: Specific murine framework mutations in h12F3 variable heavy chain variants

vH variant 30 37 48 49 73 78 93 %people HkDJ 94.0 HkDJ T N 92.0 HkDJ T L A N 90.0 hhD T N L A 90.0 HkDJ T L A N L A 88.0 HkDJ T V L A N L A 88.0 HkDJ T V L A N L A 88.0 HkDJ T V L A N L A 87.0

Table 7: Humanizing mutations in h12F3 variable kappa (vL) light chain variants

Table 8: Specific murine framework mutations in h12F3 variable kappa light chain variants

The antibodies designated as HAL1 (an antibody having a heavy chain variable region designated as vHA and a light chain variable region designated as vL1), the antibodies designated as HAL2 (an antibody having a heavy chain variable region designated as vHA and a light chain variable region designated as vL2), the antibodies designated as HAL3 (an antibody having a heavy chain variable region designated as vHA and a light chain variable region designated as vL3), the antibodies designated as HALA (an antibody having a heavy chain variable region designated as vHA and a light chain variable region designated as vLA), the antibodies designated as HALB (an antibody having a heavy chain variable region designated as vHA and a light chain variable region designated as vLB), the antibodies designated as HALC (an antibody having a heavy chain variable region designated as vHA and a light chain variable region designated as vLC), the antibodies designated as HALD (an antibody having a heavy chain variable region designated as vHA and a light chain variable region designated as v The antibodies named HALE (antibodies having a heavy chain variable region named vHA and a light chain variable region named vLE), the antibodies named HALE (antibodies having a heavy chain variable region named vHA and a light chain variable region named vLE), the antibodies named HALF (antibodies having a heavy chain variable region named vHA and a light chain variable region named vLF), the antibodies named HALG (antibodies having a heavy chain variable region named vHA and a light chain variable region named vLG), the antibodies named HALH (antibodies having a heavy chain variable region named vHA and a light chain variable region named vLH), and the antibodies named HALI (antibodies having a heavy chain variable region named vHA and a light chain variable region named vLI) can be used in the present invention to replace the HGLF antibody. Similarly, antibodies having a heavy chain variable region designated as vHA, vHB, vHC, vHD, vHE, vHF, vHG or vHH and a light chain variable region designated as vL1, vL2, vL3, vLA, vLB, vLC, vLD, vLE, vLF, vLG, vLH or vLI can be used in the present invention to replace the HGLF antibody. See Figures 5 to 8 for the sequences of vHA, vHB, vHC, vHD, vHE, vHF, vHG, vHH, vL1, vL2, vL3, vLA, vLB, vLB-Q, vLB-V, vLC, vLD, vLE, vLF, vLG, vLH and vLI.

Humanized antibodies of low quality, low expression yield, or unfavorable sequence were not evaluated in functional assays. The apparent affinity of humanized antibodies for cells expressing ALPPL2 was evaluated using flow cytometry. Briefly, the _KD of each resulting antibody was then determined by saturation binding assay. HEK293 cells stably expressing human ALPPL2 were aliquoted into 96-well v-bottom plates at 1E5 cells per well. Each humanized ALPP/ALPPL2 antibody was added at concentrations of 0.2, 2, and 20 nM and incubated on ice for 60 minutes. The cells were pelleted and washed twice with PBS/FBS, and then 10 μg/ml of APC-labeled anti-human IgG mouse secondary antibody was added and incubated on ice for another 60 minutes. The cells were pelleted and washed twice with PBS/FBS and resuspended in 100 μL 2% paraformaldehyde. Fluorescence was analyzed by flow cytometry, and the percentage of saturated fluorescence signal was used to determine the binding percentage and then calculate the apparent _KD based on the three antibody concentrations. The apparent K _D of recombinant humanized anti-ALPP/ALPPL2 was compared with c12F3 (chimeric 12F3 IgG1k) as shown in Table 9.

Table 9: Binding of hALPP-1 antibody variants on HEK-ALPPL2 cells determined by flow cytometry (KD (nM)); NT = not tested.

Example 4: h12F3 conjugation and in vitro cytotoxicity

Several h12F3 antibodies were constructed using the hIGHV3-49/hIGHJ4 heavy chain variable region human germline and the hIGKV1-33/hIGKJ 2 or hIGKV1D-43/hIGKJ2 or hIGKV1-16/hIGKJ2 light chain variable region human germline as human acceptor sequences. These antibodies differed in the amino acid residues selected for mutation back to the mouse antibody or mouse germline sequence.

As described above, humanized antibodies of low quality, low expression or unfavorable sequence were not evaluated in functional assays. For drug delivery evaluation, various humanized forms of h12F3 antibodies were conjugated to 8 loaded MDpr-PEG (12) -gluc-MMAE Auristatin T. After selecting potential antibody leads, additional cytotoxicity evaluations were performed on different payloads, including conjugation with 4 loaded mc-vc-PABC-MMAE or mp-dLAE-PABC-MMAE or antibodies conjugated with 8 loaded MDpr-PEG (12) -gluc-MMAE. The conjugation method is described in U.S. Publication No. 2018/0092984. For mp-dLAE-PABC-MMAE linker conjugation, antibody drug conjugates were prepared using the humanized anti-ALPP/ALPPL2 antibodies described herein as described in PCT/US2020/051648 (filed on September 18, 2020). For antibody conjugation with mp-dLAE-PABC-MMAE, the antibody was partially reduced using an appropriate equivalent of TCEP (tris(2-carboxyethyl)phosphine) according to the procedure of US2005/0238649, which is expressly incorporated herein by reference. Briefly, the antibody in phosphate buffered saline containing 2mM DTPA (pH 7.4) was treated with 2.1 equivalents of TCEP and then incubated at 37°C for about 45 minutes. The thiol/Ab value was checked by reacting the reduced antibody with compound 1 and determining the loading using hydrophobic interaction chromatography.

Using the method of US2005/0238649, the tripeptide-based auristatin drug-linker mp-dLAE-PABC-MMAE compound was conjugated to a partially reduced antibody, which is expressly incorporated herein by reference. In brief, the drug-linker compound (mp-dLAE-PABC-MMME) in DMSO (50% excess) was added to the reduced antibody and additional DMSO in PBS containing EDTA to obtain a total reaction co-solvent of 10%-20%. After 30 minutes at ambient temperature, an excess of QuadraSil MPTM was added to the mixture to quench all unreacted maleimide groups. The resulting ADC was then purified and the buffer was replaced by desalting into PBS buffer using Sephadex G25 resin and kept at -80°C until further use. The protein concentration of the resulting ADC composition was determined at 280nm. The drug-antibody ratio (DAR) of the conjugate was determined by hydrophobic interaction chromatography (HIC).

For in vitro cytotoxicity assays, tumor cell lines were plated 24 hours before ADC treatment. Cells were treated with a specified dose of ADC and incubated at 37 ° C for 96 hours. In some experiments, non-antigen binding ADC was included as a negative control. Cell viability of cell lines was measured using Cell Titer Glo (Promega Corporation, Madison, WI) according to the manufacturer's instructions. Cells were incubated with Cell Titer Glo reagent at room temperature for 30 minutes, and luminescence was measured on an Envision microplate reader (Perkin Elmer, Waltham, MA). As shown in Figure 9, humanized forms of the h12F3 antibody containing light chain F variants identified variants with high drug delivery capabilities, especially when compared with other combinations.

Based on cytotoxic potency and apparent affinity, the selected humanized antibodies were further evaluated for their ability to deliver different payloads to tumor cells. Humanized 12F3 antibodies with high drug delivery capacity were conjugated with 4 loaded mc-vc-MMAE or mc-vc-PABC-MMAE or with 8 loaded MDpr-PEG(12)-gluc MMAE as described previously.

Tumor cells were incubated with each ADC at 37°C for 96-144 hours. Non-binding (referred to as h00 or IgG) ADC was used as a negative control. Cell viability was measured using Cell Titer Glo according to the manufacturer's instructions. Fluorescence signals were measured on a Fusion HT fluorescent microplate reader (Perkin Elmer, Waltham, MA). Data were normalized to untreated cells and IC50 values were calculated using Graph Pad software. The results are reported in Table 10 as IC ₅₀ , i.e., the concentration of compound required to reduce viability by 50% compared to vehicle-treated cells (control = 100%). The h12F3 ADC achieved single-digit and double-digit ng/ml IC ₅₀ values in a group of cell lines with ALPP expression ranging from 30,000 to 500,000.

Table 10: IC50 (ng/ml) of h12F3 HGLF antibody drug conjugates against various cancer cells. Results are reported as IC50 and percent viability remaining at endpoint.

The cytotoxic potency of the antibody drug conjugates using humanized variants of the same payload (mp-dLAE-MMAE) was compared by plotting the IC50 values for multiple cell lines. The humanized variants of the 12F3 antibody showed similar potency in vitro as shown in FIG. 10 .

Based on its (i) binding properties, (ii) drug delivery ability, and (iii) number of back mutations compared to other variants, the antibody named HGLF (heavy chain variable region (vHG) as shown in SEQ ID NO: 15 and light chain variable region (vLF) as shown in SEQ ID NO: 30) was finally selected as the lead humanized anti-ALPP/ALPPL2 antibody (see Tables 5 to 8).

The evaluation of HGLF ADC on tumor cancer cell spheroids was performed as follows: 100uL of cells were plated at 2.5E4 cells/well in an ultra-low attachment round-bottom 96-well plate (Corning, Corning, NY) at 37°C for 48 hours. After incubation, 100uL of culture medium containing 2X ADC was added and incubated at 37°C for 120 hours. In some experiments, non-antigen binding ADC was included as a negative control. Cell viability of cell lines was measured using 3D Cell Titer Glo (Promega Corporation, Madison, WI) according to the manufacturer's instructions. Cells were incubated with 3D Cell Titer Glo reagent at room temperature for 30 minutes and luminescence was measured on an Envision microplate reader (Perkin Elmer, Waltham, MA). The results are reported as IC50, i.e., the concentration of compound required to produce a half-maximal reduction in viability compared to vehicle-treated cells (control = 100%). As shown in Figure 11 and Table 11, h12F3 HGLF ADC conjugated with vcMMAE-, mp-dLAE-MMAE-, and mdpr-gluc-MMAE-based linkers exhibited high cytotoxicity against 3D spheroids with potency similar to that in 2D cultures.

Table 11: IC50 (ng/ml) values of h12F3 HGLF ADC against tumor cell 3D spheroids

Cell lines vc-MMAE(4) dLAE-MMAE(4) MDpr-PEG(12)-gluc-MMAE RMUGS 1.6 1.1 1.0 NCI-N87 19.4 19.8 22.9

Example 5: Antibody internalization

Internalization experiments were performed on RMUGS, Hep2 parental, and Hep2 ALPP knockout cell lines by automated fluorescence microscopy (IncuCyte S3, Essen Bioscience). Cells were seeded in 96-well flat-bottomed transparent black tissue culture treated microplates (Corning, Corning, NY) and left to adhere overnight at 37°C. h12F3HGLF and non-targeting control antibodies were labeled with IncuCyte FabFluor-pH Red antibody labeling reagent (Essen Bioscience, Ann Arbor, MI) according to the manufacturer's protocol. The required volumes of test antibody, FabFluor reagent, and culture medium were calculated at 2 times the final assay concentration, and the FabFluor reagent was added at a molar ratio of 1:3 to the antibody. The antibody and FabFlour were gently mixed and incubated at 37°C for 15 minutes, and then the antibody-FabFluor complex was added to each appropriate well of the plate containing the cells. The final concentration of h12F3 HGLF and non-binding control antibody per well was 250ng/mL. The plates were arranged on a microplate tray in IncuCyte S3 (Essen Bioscience, Ann Arbor, MI) and scanned using the Adherent Cell-by-Cell protocol. Phase data and red channel data (acquisition time set to 400ms) were collected, 4 images per well, at least once every 0.5-2 hours, for up to 20 hours, and the objective lens was set to 10x. Fluorescence signal intensity was quantified using the IncuCyte software analysis tool. The analysis of each cell line was refined and adjusted using label-free cell counting and manual image selection for preview and algorithm training. After the analysis was completed, the data was plotted using the IncuCyte software, where the graph metric was set to the red mean intensity object average of each cell normalized to the non-binding control. As shown in Figure 12, h12F3HGLF is internalized in cells expressing ALPP, and internalization is specific because ALPP knockout HEP2 cells do not internalize naked antibodies.

Example 6: Kinetic Binding and pH Sensitivity

Bivalent affinity was measured by biolayer interferometry (BLI) on an Octet Red384 system (ForteBio) with an anti-human Fab-CH1 second generation (FAB2G) biosensor at pH 7.4 and 37°C. Soluble human ALPP-Fc and ALPPL2-Fc fusion dimer proteins were produced in CHO cells for use as analytes. Antibodies h12F3 HGLF and HFLD were immobilized on the biosensor at 3ug/mL for 600 seconds, and then the titrated analytes hALPP and hALPPL2 were bound at 6 concentrations ranging from 0.12-125nM for 600 seconds, followed by a final 50-minute dissociation step in kinetic buffer (1% casein and 0.2% Tween20 in 1× PBS pH7.4). After subtracting the reference of the probe-only curve, the data were globally fitted with a 1:1 model without Rmax sensor connection. Using curve fitting at concentrations of 31.3, 7.8, 1.95, 0.49, and 0.12 nM, the bivalent binding of h12F3 HGLF to hALPP and hALPPL2 was measured to be 1.3E-10 M (k _d 2.0E-05 1/s/ _ka 1.5E51/Ms) and 4.4E-11 M (k _d 7.1E-06 1/s/ _ka 1.6E5 1/Ms), respectively. Compared to HGLF, the affinity of the HFLD variant for hALPP and hALPPL2 was reduced by 26.9-fold and 34-fold, respectively, as shown in Figure 13.

To assess pH sensitivity, the bivalent affinity at pH 6.0, 37°C was determined using the same BLI method as the pH 7.4 experiment. The only difference was the use of a different kinetic buffer (1% casein and 0.2% Tween20 in phosphate citrate, pH 6.0). Using 800 seconds of dissociation and curve fitting at concentrations of 125, 31.3, 7.8, 1.95, 0.49, and 0.12 nM, the bivalent binding of h12F3 HGLF to hALPP and hALPPL2 was measured to be 6.8E-09 M (k _d 5.9E-04 1/s/ _ka 8.7E4 1/Ms) and 4.8E-9 M (k _d 4.3E-04 1/s/ _ka 8.9E4 1/Ms), respectively. As shown in Figure 14, compared to pH 7.4 affinity, hALPP Fc was reduced 52-fold, and hALPPL2 was reduced 109-fold.

Example 7: In vivo antitumor activity

NSG mice were inoculated subcutaneously with 5×10 ⁵ CAOV3p1 or 2.5×10 ⁶ NCI-H1651 cells. Nude mice were inoculated subcutaneously with 1×10 ⁷ NCI-N87, 2×10 ⁶ RMUG-S, 1×10 ⁷ LoVo, and 5×10 ⁶ HT-1376 cells. According to the manufacturer's regulations, 0.1 ml of PBS and Matrigel (1:1) were inoculated subcutaneously in the right flank of each mouse. Tumor growth was monitored with a caliper, and the average tumor volume was calculated using the formula (0.5×[length× ^width2 ]). When the average tumor volume reached approximately 100-200 mm ³ , the mice were randomly divided into different groups, including untreated conditions or intraperitoneal injections of h12F3 HGLF or HFLD conjugated with mp-dLAE-MMAE or vcMMAE, four times every four days (q4d×4) or three times every 7 days (q7d×3). When the tumor volume reached approximately 800-1000 ^mm3 , the mice were euthanized. %TGI was defined as (1-(mean volume of treated tumors)/(mean volume of control tumors)) × 100%. All animal procedures were performed in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited institution in accordance with protocols approved by the Institutional Animal Care and Use Committee.

For the ovarian tumor model CAOV3, the resulting tumor volumes over time for untreated mice and mice treated with 3 and 5 mg/kg h12F3 HGLF or HFLD-dLAE-MMAE are shown in Figure 15. For the gastric tumor model NCI-N87, the resulting tumor volumes over time for untreated mice and mice treated with 1 and 3 mg/kg h12F3 HGLF or HFLD-dLAE-MMAE are shown in Figure 16. For the gastric tumor model NCI-N87, the resulting tumor volumes over time for untreated mice and mice treated with 3 mg/kg h12F3 ADC conjugated with vc-MMAE and dLAE-MMAE are shown in Figure 17. The ALPP ADC showed similar antitumor activity in vivo.

For the lung tumor model NCI-H1651, the resulting tumor volumes over time for untreated mice and mice treated with 3 mg/kg h12F3 ADC conjugated with vc-MMAE and dLAE-MMAE are shown in Figure 18. The ALPP ADC showed similar anti-tumor activity in vivo. The resulting tumor growth inhibition in seven xenograft models is shown in Figure 19. The bar graph summarizes the % tumor volume change of the treatment group relative to the control. The comparison was made at 3 mg/kg h12F3-HFLD ADC conjugated with vc-MMAE and dLAE-MMAE. The average anti-tumor activity of the non-binding ADC control is shown by the dotted line.

In another set of assays, NSG mice were subcutaneously inoculated with 5×10 ⁵ CAOV3p1, NCG mice were subcutaneously inoculated with 5×106 SNU-2535, nude mice were subcutaneously inoculated with 1×10 ⁷ NCI-N87, and SCID mice were subcutaneously inoculated with 1×10 ⁷ HPAC. According to the manufacturer's specifications, 0.1 ml of PBS and Matrigel (1:1) were subcutaneously inoculated in the right flank of each mouse. Tumor growth was monitored with a caliper, and the average tumor volume was calculated using the formula (0.5×[length× ^width2 ]). When the average tumor volume reached approximately 100-200 mm ³ , the mice were randomly divided into different groups, including untreated conditions or intraperitoneal injections of h12F3HGLF conjugated with mp-dLAE-MMAE or mc-vc-MMAE, four times every 4 days (q4d×4) or three times every 7 days (q7d×3). When the tumor volume reached approximately 2-3000 mm ³ , the mice were euthanized. %TGI was defined as (1-(mean volume of treated tumors)/(mean volume of control tumors)) x 100%. All animal procedures were performed in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited institution according to protocols approved by the Institutional Animal Care and Use Committee.

For the gastric tumor model NCI-N87, the resulting tumor volumes over time for untreated mice and mice treated with 1 or 3 mg/kg h12F3 ADC conjugated to vc-MMAE and dLAE-MMAE are shown in Figure 20. The ALPP ADCs showed similar anti-tumor activity in vivo.

For the pancreatic tumor model HPAC, the resulting tumor volumes over time for untreated mice and mice treated with 1 or 3 mg/kg h12F3 ADC conjugated with mc-vc-MMAE and mp-dLAE-MMAE are shown in Figure 21. ALPP ADC showed similar anti-tumor activity in vivo. The resulting tumor growth inhibition in the four xenograft models is shown in Figure 22. The bar graph summarizes the percentage change in tumor volume of the treatment group relative to the control. The comparison was made at 3 mg/kg h12F3-HGLF ADC conjugated with vc-MMAE and dLAE-MMAE. The average anti-tumor activity of the non-binding ADC control is shown by the dotted line.

In another assay, twelve patient-derived xenografts were tested for antitumor activity in nude mice using a 2+2 experimental design. In brief, when tumors of sufficient reserve animals reached 1.0-1.5 cm ³ , tumors were harvested to be re-implanted into the animals before the study. Pre-study animals were implanted with tumor fragments harvested from reserve animals unilaterally on the left flank. Each animal was implanted and recorded from a specific passage batch. When the tumor reached an average tumor volume of 150-300 mm ³ , the animals were matched to the treatment group or control group for administration by tumor volume and administration was started on day 0. The h12F3-mc-vc-MMAE conjugate was administered at 5 mg/kg (QWx3) and compared with the PBS-treated cohort. Tumor volume was measured twice a week. Final tumor volume measurements were performed on the day the study reached the endpoint. Starting on day 0, tumor size was measured twice a week by digital calipers, and data for each group were recorded, including individual and average estimated tumor volumes (mean TV ± SEM); tumor volume was calculated using formula (1): TV = width 2 × length × 0.52. At the completion of the study, the percent tumor growth inhibition (%TGI) value for each treatment group (T) versus control (C) was calculated and reported using the initial (i) and final (f) tumor measurements by formula (2): %TGI = 1-(Tf-Ti)/(Cf-Ci). As shown in Figure 23, the h12F3-mc-vc-MMAE conjugate SGN-ALPV showed anti-tumor activity in 58% (7/12) of PDX models with heterologous target expression. At the doses used, the response models exhibited TGIs ranging from 55% to >100%. Anti-tumor activity was observed in PDX models from patients who had received chemotherapy pretreatment and those who had not received treatment (Figure 23, B and C).

Example 8: Cross-reactivity and epitope mapping

To confirm the cross-reactivity of the antibodies with the orthologous ALPP protein, the cynomolgus macaque ( macacafalsicularis ) ALPP gene (NHP ALPP) was transfected into HEK293 cells and the antibodies were screened by flow cytometry. Briefly, the KD of each resulting antibody was then determined by saturation binding assay. 1×105 HEK293 cells stably expressing human ALPPL or ALPPL2 and NHP ALPP were aliquoted into each well of a 96-well v-bottom plate. h12F3 HGLF and HFLD antibodies were added at concentrations ranging from 0.2nM to 20nM and incubated on ice for 60 minutes. The cells were pelleted and washed 3 times with PBS/BSA, and then 10ug/ml of APC-labeled anti-human IgG goat secondary antibody was added and incubated on ice for another 60 minutes. The cells were pelleted and washed 3 times with PBS/BSA and resuspended in 125μL PBS/BSA. Fluorescence was analyzed by flow cytometry and the percentage of saturated fluorescent signal was used to determine the percent binding and subsequently calculate the apparent KD. The apparent _KD for both antibodies is shown in Figure 24. Importantly, although the antibody variant h12F3 HFLD had a significantly reduced affinity for the monkey ortholog, the HGLF variant exhibited similar binding characteristics for both human and monkey placental alkaline phosphatase.

Since h12F3 HGLF does not cross-react with rat ALPP/ALPPL2 orthologs, the human ALPP region was exchanged with the homolog region from rat ALPP. These constructs were transiently transfected into 2×10 ⁶ cells HEK293 cells using lipofectamine 3000 (1:1.5 DNA/lipofectamine ratio) according to the manufacturer's instructions. The h12F3 HGLF binding epitope was assessed on cells expressing chimeric rat/human ALPP variants 48 hours after transfection using flow cytometry as described previously. As shown in Figure 25, when the human ALPP region containing aa L287-S339 was replaced by the rat ALPP sequence, h12F3 HGLF binding was impaired.

Example 9: Kinetic binding of antibodies to Fc receptors

Antibody-based immune responses are driven by interactions with Fc receptors on immune cells. Therefore, to determine the ability of h12F3 HGLF, h12F3 HGLF-mc-vc-MMAE, or h12F3HGLF-mp-dLAE-MMAE to interact with Fc receptors, binding kinetics to hFcγRI, hFcγRIIa H131, hFcγRIIa R131, hFcγRIIIa F158, hFcγRIIIa V158, and hscFcRN were assessed by biolayer interferometry (BLI). Biotinylated avi-tagged human Fc receptors fused to monomeric Fc (designed and expressed at Seagen) were loaded onto high-precision streptavidin biosensors (from ForteBio), and all receptors responded around 0.4 nm, except for hFcγR1, which responded around 1.2 nm. An initial baseline was completed in immobilization buffer (0.1% BSA, 0.02% Tween 20, 1x PBS pH 7.4), followed by a second baseline in kinetic buffer (1% casein, 0.2% Tween 20, 1x PBS pH 7.4 for hFcγRI, IIa, IIIa and IIb interactions, 1% BSA + 0.2% Tween 20, phosphate citrate pH 6.0 for hscFcRN interactions). Titrated h12F3 HGLF, h12F3HGLF-mc-vc-MMAE, h12F3 HGLF-mp-dLAE-MMAE, and positive control mAb samples were bound and dissociated as follows: 600 s and 1000 s for hFcγRI, 10 s and 50 s for hFcγRIIa and hFcγRIIb, 60 s and 200 s for hFcγRIIIa, and 50 s and 200 s for hscFcRN in kinetic buffer. Sensorgrams were generated on an Octet HTX system (ForteBio) at 30 °C and globally fit with a 1:1 kinetic Langmuir isotherm model (Rmax not connected) after subtracting the reference of the antigen-loaded 0 nM analyte sensor. A negative control with the highest concentration of antibody and ADC without immobilized Fc receptor (20 μM) was also included to verify the absence of nonspecific binding of the analyte to the streptavidin biosensor itself. The specific loading concentration and time of each receptor of the streptavidin sensor and the concentration of the titrated analyte are listed (Table 12 and Table 13). In summary, the parental antibody and mc-vc-MMAE ADC bind to all human Fc receptors, as shown in Figure 26. The affinity for hFcγRI is the highest, ranging from about 1.3-2.2nM, while the affinity for hFcRN is second, at about 10.6-13.9nM. The affinities of hFcγRIIa and hFcγRIIIa variants range from 0.81-7.3μM, while hFcγRIIb shows the weakest affinity range of 36-67μM. Compared with the positive control mAb results, the affinities of h12F3 HGLF-mc-vc-MMAE and h12F3 HGLF-mp-dLAE-MMAE for all human Fc receptors are very similar and comparable to the parental antibody h12F3 HGLF.

Table 12: Immobilization concentration and time on streptavidin biosensor

Table 11: Analyte concentrations

1.- For 12F3HGLF-based conjugates using mc-vc-MMAE and mp-dLAE-MMAE, use the same concentration

Antibody-dependent cellular cytotoxicity (ADCC) of primary NK cells

To determine whether the h12F3 HGLF backbone and the derived conjugates induce antibody-dependent cellular cytotoxicity (ADCC), cells expressing ALPPL2 were incubated with natural killer (NK) cells in vitro in the presence of h12F3 HGLF, h12F3 HGLF-mc-vc-MMAE or h12F3 HGLF-mp-dLAE-MMAE. After incubation, the percentage of cell lysis was measured. In brief, effector cell preparation: peripheral blood mononuclear cells (PBMCs) were rapidly thawed in a 37°C water bath one day before the assay. The cells were transferred to a 50 mL tube containing AIM-V medium (Gibco, catalog number 12055091) supplemented with 5% heat-inactivated human serum (Gemini Bio-products, catalog number 100-512) (AIM-V/5% HIHS). The cells were centrifuged at 1500 rpm for 10 minutes. PBMCs were resuspended in AIM-V/5% HIHS (1:50 dilution with 1mg/mL stock solution) containing DNase I (Sigma-Aldrich, catalog number D5025) at a final concentration of 20 μg/mL and incubated at 37°C for 10 to 15 minutes. Cells were centrifuged and resuspended in AIM-V/5% HIHS as above. Cells were counted and seeded in T150 flasks at a concentration of 2-4×108 cells/flask, 25mL per flask. Cells were incubated overnight undisturbed at 37°C, 5% CO2, in a humidified incubator. The next day, non-adherent cells were collected and the flasks were rinsed vigorously 3 times (7mL) with PBS. The rinse solution was merged with the non-adherent cells and precipitated by centrifugation at 1500rpm for 7 minutes. Resuspend the cells in a small volume (2 mL) for counting, and adjust the cell suspension to a concentration of 5×107 cells/mL in PBS+2% FBS (as recommended by the EasySep protocol). Isolate NK cells by negative selection according to the EasySep Human NK Cell Enrichment Kit (Stem Cell Technology, Catalog No. 19055) instructions. The enriched effector cells are then suspended in RPMI/1% FBS at a concentration of 7.2×105 cells/mL (so that 70 μL contains approximately 5×104 effector cells). Target cell preparation: LoVo cells expressing ALPPL2 are collected and counted. Next, 5×106 cells are removed and precipitated by centrifugation. Resuspend the cells in 100 μL of FBS. Then, 100 μL (approximately 100 μCi) Cr-51 (Perkin Elmer Health Sciences, Inc., Catalog No. NEZ030S) is added to the cells and mixed by gentle tapping. The cells were placed in a 37°C, 5% CO2, humidified incubator for 1 hour, occasionally tapping the tube to suspend the cells. Wash the cells 3 times with RPMI/1% FBS. Tap the tube between washes to loosen the cell pellet. After washing, the cells were resuspended in 10mL RPMI/1% FBS and counted. Then, 7.2×105 cells were removed and suspended in a total volume of 10mL of assay medium, so that 70μL was equivalent to ~5×103 target cells. Preparation of ADC and antibody diluents and plate assembly: Antibodies and ADCs were diluted in assay medium at 3x concentration. The antibodies tested were h12F3 HGLF, h12F3 HGLF-mc-vc-MMAE or h12F3 HGLF-mp-dLAE-MMAE, afucosylated antibody that binds to CD71, and isotype controls. Antibodies were added to the assay plate before adding Cr-labeled target cells. In addition, 70 μL and 140 μL of assay medium were added to the control wells representing the total release control and spontaneous release control, respectively, instead of the antibody. Finally, the target cells were mixed and 70 μL was added to each test well and control well of the 96-well plate. The target was incubated with mAb at 37°C, 5% CO2, in a humidified incubator for 30 minutes. Then, 70 μL (5×104) of effector cells were added to each test well, while 70 μL of 3% Triton X-100 was added to the total release well and mixed. The plate was returned to a 37°C, 5%, CO2, humidified incubator for 4 hours. After incubation, 35 μL of supernatant was transferred to the Luma plate. The Luma plate was dried overnight, then covered with sealing tape and read on a Perkin Elmer TopCount NXT microplate scintillation counter. The analysis was performed by calculating the % specific lysis as follows (analyzed with GraphPad Prism): % specific lysis = [(test cpm - background cpm) ÷ (total cpm - background cpm)] x 100. As shown in Figure 27, NK cell cytotoxicity was mediated in vitro in the presence of h12F3 HGLF antibody as well as h12F3 HGLF-mc-vc-MMAE and h12F3 HGLF-mp-dLAE-MMAE. This activity was similar to the positive control and was mediated by the presence of the target on the cells since the non-binding antibody could not stimulate the effector cells.

Antibody-dependent cellular cytotoxicity

To determine whether h12F3 HGLF, h12F3 HGLF-mc-vc-MMAE or h12F3HGLF-mp-dLAE-MMAE showed ADCP activity, fluorescent cells coated with antibodies or ADCs expressing ALPP/ALPPL2 were co-incubated with primary macrophages and phagocytosis was measured by fluorescent flow cytometry. In brief, LoVo tumor cells were fluorescently labeled with PKH26 according to the manufacturer's instructions. Cells were harvested from culture dishes with 0.05% trypsin EDTA and washed once with 1xPBS. The cells were resuspended in 1 mL of diluent C contained in the PKH26 red fluorescent cell membrane labeling kit (Sigma-Aldrich, catalog number PKH26GL-1KT). In a separate test tube, 1 mL of diluent C + 4 μL PKH26 dye was added and pipetted up and down to mix. The staining solution was transferred to the resuspended cells and quickly mixed by pipetting up and down several times. The cells were incubated at room temperature for 5 minutes and the labeling reaction was terminated by adding 2 mL FBS. The cells were washed 3 times with RPMI/10% FBS and resuspended in PBS at a concentration of 0.8×106 cells/mL. The labeled target cells were transferred to a 96-well U-shaped bottom plate and treated with test antibodies, ADCs or isotype control antibodies using the following steps. In a separate 96-well U-shaped bottom plate, 10 times the stock solution of mAb, ADC and isotype control in PBS was diluted in PBS at 1:10, and 33 μL/well was added to the appropriate wells of the cells in the U-shaped bottom plate. The plate was incubated at room temperature for 30 minutes, centrifuged, and washed once with 200 μL/well culture medium (RPMI/10% FBS). The cells were resuspended in 330 μL/well culture medium (RPMI/10% FBS). One day before the assay, PBMCs from 2 healthy donors were thawed in a 37°C water bath and transferred to RPMI/10% FBS (0.1-0.2EU/mL). A total of 0.7 × 106 PBMCs per well were added to a 48-well flat-bottom plate and allowed to adhere overnight. The old culture medium (and non-adherent cells) was aspirated and replaced with 200 μL of fresh culture medium. Next, 100 μL of labeled, treated target cells in each well were transferred in triplicate to the corresponding wells of the adherent monocyte/macrophage flat-bottom plate, and the plate was incubated overnight at 37 ° C for 16-18 hours. All cells in the 48-well plate were harvested by collecting the sample solution, collecting the wash solution with 1x PBS and separating with 1x Versene. Macrophages were fluorescently labeled using the following steps: target cells and macrophages were collected in a U-shaped bottom plate, centrifuged, resuspended in 50 μL FACS staining buffer containing human Fc fragment blocking agent (1:20 dilution), and incubated on ice for 30 minutes. Next, 50 μL of 1:50 dilutions of CD14-BV421 and CD45-APC-Cy7 antibodies diluted in FACS staining buffer were added to each well and incubated on ice for 30 minutes in foil. The cells were centrifuged, washed twice with FACS buffer, and resuspended in 1x PBS for subsequent flow cytometry analysis on an Attune NxT flow cytometer. FlowJo was used to analyze the YL1 GeoMean fluorescence (MFI of CD14+/CD45+ cells) of CD14+/CD45 cells, and the values were then exported to Excel and GraphPad Prism for further data analysis. Phagocytosis is reported as the MFI of CD14+ cells. As shown in Figure 28, the presence of h12F3 HGLF, h12F3HGLF-mc-vc-MMAE or h12F3 HGLF-mp-dLAE-MMAE conjugates enables phagocytosis of target expressing cells with similar kinetics to the positive control (anti-CD47 antibody). This activity was dependent on the presence of ALPPL2 expression on target cells, as non-binding antibodies did not induce any cell death.

In another assay, FcgRIII-dependent antibody-dependent cellular cytotoxicity (ADCC) was measured by using Promega's alternative luciferase-mediated bioassay. Briefly, cells expressing ALPP/ALPPL2 were plated on 96-well plates and co-cultured with ADCC bioassay effector cells (Promega) in the presence of increasing amounts of naked h12F3 antibodies or h12F3 HGLF antibodies conjugated to 4 or 8 mc-vc-PABC-MMAE or MDpr-PEG(12)-gluc-MMAE molecules, respectively. 24 hours after treatment, cells were incubated with Bio-Glo (Promega) according to the manufacturing method and luminescence was measured using the Envision platform. As shown in Figure 29, h12F3HGLF was able to activate FcgRIII signaling in the reporter cell line with similar kinetics to the mc-vc-PABC-MMAE conjugated to the h12F3 HGLF ADC. Conjugation with eight MDpr-PEG(12)-gluc-MMAE molecules reduced ADCC activity compared to naked h12F3 HGLF.

Example 10: Immunogenic cell death

Activation of signaling pathways for immunogenic cell death

To determine whether h12F3 HGLF-mc-vc-MMAE and h12F3 HGLF-mp-dLAE-MMAE can activate the hallmarks of ICD, cells expressing ALPP/ALPPL 2 were treated with ADC, and the phosphorylation status of IRE and JNK pathways was determined using immunoblotting. Briefly, four million LOVO cells were plated in 10 mL of competitive growth medium (Ham's F-12K (Kaighn's) medium + 10% FBS) in a 10 cm TC-treated culture dish and allowed to adhere overnight. Cells were treated with 10 nM MMAAE or 1 and 10 ug/mL of h12F3 HGLF-mc-vc-MMAE or h12F3 HGLF-mp-dLAE-MM AE in complete medium. The cells were then incubated in the treated medium for 48 or 96 hours. Cells were collected, washed, resuspended in 500 uL cold PBS and transferred to an Eppendorf tube. The samples were spun at 300xg and 4 degrees for 3 minutes. The supernatant was removed and the cells were resuspended in RIPA lysis buffer containing proteases and phosphatases. After incubation on ice for 5 minutes, the samples were spun at 17000xg and 4 degrees for 10 minutes, the supernatant was collected, and stored at -80°C. The quantitative samples were separated in NuPAGE 4%-12% bis-tris gels and run in MES running buffer for smaller proteins or in MOPS for larger proteins (165v 40 minutes). The gel was transferred to a PVDF membrane using iBlot2 (20v, 7 minutes). The membrane was briefly rinsed in DI water and then placed in blocking buffer (TBS+0.1% tween-20+5% BSA) overnight at 4 degrees. The blot was then incubated with a primary antibody for IRE, JNK, p-IRE or p-JNK at a dilution of 1:1000 in blocking buffer at room temperature for 2 hours. p-ERK was used at 1:500 and incubated in the same manner. The blots were washed 3 times with TBST (TBS + 0.1% Tween-20). Anti-rabbit peroxidase secondary antibody was prepared at a dilution of 1:10,000 in blocking buffer. The blots were incubated with the secondary antibody for 1 hour at room temperature. The blots were washed again 3 times with TBST. The blots were analyzed using SignalFire ECL and imaged on AmershamImager 600. The blots were then stripped and re-probed for GAPDH as a loading control and blotted as described above. As shown in Figure 30, incubation of LoVo cells with h12F3 HGLF-mc-vc-MMAE or h12F3 HGLF-mp-dLAE-MMAE increased the phosphorylation levels of pIR E and pJNK, which play a key role in the activation of immunogenic cell death processes.

To determine whether treatment with the h12F3 HGLF ADC lead resulted in ATP release in the culture medium, 600,000 LOVO cells were plated in 2 mL of competitive growth medium (Ham's F-12K (Kaighn's) medium + 10% FBS) in a 6-well TC-treated culture dish and allowed to adhere overnight. Solutions of 10 nM MMAE, 1 or 10 μg/mL h12F3 HGLF mp-dLAE-MMAE or mc-vc-MMAE were prepared in complete culture medium. The cells were then incubated in the treated culture medium for 24, 48 or 72 hours. At each endpoint, 500 uL of supernatant was collected from each well (sample). The supernatant was spun at 200 x g, 4 degrees for 1 minute to carefully remove any cell debris. Three 50 uL aliquots of each supernatant (for triplicate data) were then placed in a black-walled, clear-bottomed 96-well plate. Then add 50uL of reconstituted Cell Titer Glo to all wells containing supernatant. Cover the plate and protect from light. Then read the plate on the Envision microplate reader. The raw luminescence data of the triplicate data of all samples were averaged. To determine the fold change compared to the untreated sample, the average luminescence of the experimental sample was divided by the average of the untreated sample. As shown in Figure 31, h12F3 HGLF mp-dLAE-MMAE or mc-vc-MMAE conjugates all resulted in the release of ATP, which is a hallmark of immunogenic cell death.

Example 11: Pharmacokinetics

The pharmacokinetic evaluation of humanized h12F3 ADC was performed in non-human primates. Antibody drug conjugates containing h12F3 HGLF-vc-MMAE (4) and HGLF-dLAE-MMAE (4) were administered once at 1 mg/kg, and plasma samples were collected at designated time points. Total h12F3 HGLF-vc-MMAE (4) and HGLF-dLA E-MMAE (4) cynomolgus monkey plasma levels were analyzed using the Gyrolab (Gyros Protein Technologies, Sweden) 1-step universal total antibody (gTAb) assay. Briefly, assay standards and quality control samples (QC) were prepared using the administered test article diluted in pooled cynomolgus monkey K2EDTA plasma (BioIVT). Study samples with test article concentrations exceeding the assay quantitation limit were diluted to a certain range with drug-naive cynomolgus monkey K2EDTA plasma. Standards, QCs and research samples were diluted to a minimum required dilution (MRD) of 1:20 in Rexxip HX buffer (Gyros Protein Technologies, Sweden). A 30 nM equimolar master mix solution was prepared by diluting biotinylated anti-human κ light chain (Seagen) and AlexaFlour-647 anti-human Fcγ (Jackson Immunoresearch) in 1x phosphate buffered saline solution containing 0.01% (v/v) tween-20 (PBST). Equal volumes of MRD standards, QCs or research samples and master mix solutions were mixed. The resulting solution was incubated in the dark and shaken at room temperature for 1-2 hours. After incubation, the solution was transferred to a 96-well PCR plate and added to a Gyrolab Bioaffy 1000CD (Gyros Protein Technologies, Sweden), where the sample was passed through a streptavidin affinity column in the CD. The column was washed 4 times with PBST, and the associated fluorescence on the column was detected at 635 nm. Gyrolab Evaluator software was used to fit the fluorescence response of the calibrant to a 5-parameter logistic regression (5-PL). Total h12F3 HGLF-vcMMAE and HGLF-dLAE-MMAE QC and research sample concentrations were interpolated according to their respective fitted standard curves and used for pharmacokinetic evaluation. Where appropriate, Phoenix Win Nonlin (version 8.2, Certara USA, Inc.) was used to determine PK parameters by non-compartmental analysis (NCA). The following PK parameters were determined: area under the plasma concentration-time curve (AUC0-21) to 21 days, maximum plasma concentration observed (Cmax), terminal half-life, clearance (Cl), and calculated steady-state distribution volume (Vss). AUC was calculated using a linear trapezoidal linear method. For plasma concentration-time curves with adjustment R2≥0.8 and extrapolation AUC0-inf<20%, half-life, Cl, and Vss were reported. The resulting pharmacokinetic parameters are presented in Table 14, showing that the h12F3 HG LF conjugates with both vcMMAE and mp-dLAE-MMAE displayed similar antibody-conjugated MMAE pharmacokinetic profiles with no evidence of target-mediated drug disposition when compared to the non-binding ADC control.

Table 14

In order to quantify the MMAE (acMMAE) conjugated with antibodies, plasma samples were first immunocaptured at 2-8°C to separate ADC (MAbSelect, GE Healthcare) for one hour. The bound samples were washed with papain digestion buffer (20mM KPO4, 10mMEDTA, 20mM cysteine HCl), and then 2mg/mL papain in the digestion buffer was added to each sample. The samples were incubated at 37°C for four hours to enzymatically release acMMAE. Solid phase extraction was used to extract the released acMMAE. Each sample was then analyzed using normal phase UPLC (Betasil, ThermoFisher) in conjunction with tandem mass spectrometry (Sciex 6500+Triple Quad). Table 15 shows similar pharmacokinetic parameters for MMAE conjugated with antibodies using h12F3 HGLF vc-MMAE and HGLF-dLAE-MMAE conjugates, wherein the latter shows an extended half-life.

Table 15

The tolerability of the h12F3 HGLF ADC was evaluated in cynomolgus monkeys, a pharmacologically relevant species with comparable binding affinity to human and cynomolgus ALPP orthologs. Female monkeys were administered 5 mg/kg of h12F3HGLF-vc-MMAE(4) or 5, 8, 9, and 10 mg/kg of h12F3 HGLF-dLAE-MMAE(4) once a week for four weeks (q1wx4). Toxicological assessments included body weight, clinical observations, hematology, coagulation, serum chemistry, and TK. Gross pathology was performed at autopsy in the terminal phase (1 week after the last dose) and recovery phase (4 weeks after the last dose), and tissues were examined by histopathology. The maximum tolerated dose of h12F3 HGLF-vc-MMAE(4) was 5 mg/kg, and the maximum tolerated dose of h12F3 HGLF-dLAE-MMAE(4) was 9 mg/kg (Table 16). Myelotoxicity consistent with MMAE pharmacology was detected for both ADCs by hematologic and anatomic pathology assessments and was considered a dose-limiting toxicity. Other toxicities included accumulation of alveolar macrophages in the lungs, decreased numbers of secondary and tertiary follicles in the ovaries, and lymphoid depletion in the thymus.

Table 16

Incorporated by Reference

All references cited herein, including patents, patent applications, scientific papers, textbooks, etc., are incorporated by reference in their entirety.

Sequence Listing

<110> Sijin Co., Ltd.

<120> Anti-ALPP/ALPPL2 antibodies and antibody-drug conjugates

<130> 5620-00112PC

<150> US 63/162,635

<151> 2021-03-18

<150> US 63/301,574

<151> 2022-01-21

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atgctggggc cctgcatgct gctgctgctg ctgctgctgg gcctgaggct acagctctcc 60

ctgggcatca tcccagttga ggaggagaac ccggacttct ggaaccgcga ggcagccgag 120

gccctgggtg ccgccaagaa gctgcagcct gcacagacag ccgccaagaa cctcatcatc 180

ttcctgggcg atgggatggg ggtgtctacg gtgacagctg ccaggatcct aaaagggcag 240

aagaaggaca aactggggcc tgagataccc ctggccatgg accgcttccc atatgtggct 300

ctgtccaaga catacaatgt agacaaacat gtgccagaca gtggagccac agccacggcc 360

tacctgtgcg gggtcaaggg caacttccag accattggct tgagtgcagc cgcccgcttt 420

aaccagtgca acacgacacg cggcaacgag gtcatctccg tgatgaatcg ggccaagaaa 480

gcagggaagt cagtggggagt ggtaaccacc acacgagtgc agcacgcctc gccagccggc 540

acctacgccc acacggtgaa ccgcaactgg tactcggacg ccgacgtgcc tgcctccgcc 600

cgccaggagg ggtgccagga catcgctacg cagctcatct ccaacatgga cattgacgtg 660

atcctaggtg gaggccgaaa gtacatgttt cgcatgggaa ccccagaccc tgagtaccca 720

gatgactaca gccaaggtgg gaccaggctg gacgggaaga atctggtgca ggaatggctg 780

gcgaagcgcc agggtgcccg gtatgtgtgg aaccgcactg agctcatgca ggcttccctg 840

gacccgtctg tgacccatct catgggtctc tttgagcctg gagacatgaa atacgagatc 900

caccgagact ccacactgga cccctccctg atggagatga cagaggctgc cctgcgcctg 960

ctgagcagga accccgcgg cttcttcctc ttcgtggagg gtggtcgcat cgaccatggt 1020

catcatgaaa gcagggctta ccgggcactg actgagacga tcatgttcga cgacgccatt 1080

gagagggcgg gccagctcac cagcgaggag gacacgctga gcctcgtcac tgccgaccac 1140

tcccacgtct tctccttcgg aggctacccc ctgcgaggga gctccatctt cgggctggcc 1200

cctggcaagg cccgggacag gaaggcctac acggtcctcc tatacggaaa cggtccaggc 1260

tatgtgctca aggacggcgc ccggccggat gttaccgaga gcgagagcgg gagccccgag 1320

tatcggcagc agtcagcagt gcccctggac gaagagaccc acgcaggcga ggacgtggcg 1380

gtgttcgcgc gcggcccgca ggcgcacctg gttcacggcg tgcaggagca gaccttcata 1440

gcgcacgtca tggccttcgc cgcctgcctg gagccctaca ccgcctgcga cctggcgccc 1500

cccgccggca ccaccgacgc cgcgcacccg gggcggtccg tggtccccgc gttgcttcct 1560

ctgctggccg ggaccctgct gctgctggag acggccactg ctccctga 1608

<210> 2

<211> 535

<212> PRT

<213> Homo sapiens

<400> 2

Met Leu Gly Pro Cys Met Leu Leu Leu Leu Leu Leu Leu Gly Leu Arg

1 5 10 15

Leu Gln Leu Ser Leu Gly Ile Ile Pro Val Glu Glu Glu Asn Pro Asp

20 25 30

Phe Trp Asn Arg Glu Ala Ala Glu Ala Leu Gly Ala Ala Lys Lys Leu

35 40 45

Gln Pro Ala Gln Thr Ala Ala Lys Asn Leu Ile Ile Phe Leu Gly Asp

50 55 60

Gly Met Gly Val Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln

65 70 75 80

Lys Lys Asp Lys Leu Gly Pro Glu Ile Pro Leu Ala Met Asp Arg Phe

85 90 95

Pro Tyr Val Ala Leu Ser Lys Thr Tyr Asn Val Asp Lys His Val Pro

100 105 110

Asp Ser Gly Ala Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Gly Asn

115 120 125

Phe Gln Thr Ile Gly Leu Ser Ala Ala Ala Arg Phe Asn Gln Cys Asn

130 135 140

Thr Thr Arg Gly Asn Glu Val Ile Ser Val Met Asn Arg Ala Lys Lys

145 150 155 160

Ala Gly Lys Ser Val Gly Val Val Thr Thr Thr Arg Val Gln His Ala

165 170 175

Ser Pro Ala Gly Thr Tyr Ala His Thr Val Asn Arg Asn Trp Tyr Ser

180 185 190

Asp Ala Asp Val Pro Ala Ser Ala Arg Gln Glu Gly Cys Gln Asp Ile

195 200 205

Ala Thr Gln Leu Ile Ser Asn Met Asp Ile Asp Val Ile Leu Gly Gly

210 215 220

Gly Arg Lys Tyr Met Phe Arg Met Gly Thr Pro Asp Pro Glu Tyr Pro

225 230 235 240

Asp Asp Tyr Ser Gln Gly Gly Thr Arg Leu Asp Gly Lys Asn Leu Val

245 250 255

Gln Glu Trp Leu Ala Lys Arg Gln Gly Ala Arg Tyr Val Trp Asn Arg

260 265 270

Thr Glu Leu Met Gln Ala Ser Leu Asp Pro Ser Val Thr His Leu Met

275 280 285

Gly Leu Phe Glu Pro Gly Asp Met Lys Tyr Glu Ile His Arg Asp Ser

290 295 300

Thr Leu Asp Pro Ser Leu Met Glu Met Thr Glu Ala Ala Leu Arg Leu

305 310 315 320

Leu Ser Arg Asn Pro Arg Gly Phe Phe Leu Phe Val Glu Gly Gly Arg

325 330 335

Ile Asp His Gly His His Glu Ser Arg Ala Tyr Arg Ala Leu Thr Glu

340 345 350

Thr Ile Met Phe Asp Asp Ala Ile Glu Arg Ala Gly Gln Leu Thr Ser

355 360 365

Glu Glu Asp Thr Leu Ser Leu Val Thr Ala Asp His Ser His Val Phe

370 375 380

Ser Phe Gly Gly Tyr Pro Leu Arg Gly Ser Ser Ile Phe Gly Leu Ala

385 390 395 400

Pro Gly Lys Ala Arg Asp Arg Lys Ala Tyr Thr Val Leu Leu Tyr Gly

405 410 415

Asn Gly Pro Gly Tyr Val Leu Lys Asp Gly Ala Arg Pro Asp Val Thr

420 425 430

Glu Ser Glu Ser Gly Ser Pro Glu Tyr Arg Gln Gln Ser Ala Val Pro

435 440 445

Leu Asp Glu Glu Thr His Ala Gly Glu Asp Val Ala Val Phe Ala Arg

450 455 460

Gly Pro Gln Ala His Leu Val His Gly Val Gln Glu Gln Thr Phe Ile

465 470 475 480

Ala His Val Met Ala Phe Ala Ala Cys Leu Glu Pro Tyr Thr Ala Cys

485 490 495

Asp Leu Ala Pro Pro Ala Gly Thr Thr Asp Ala Ala His Pro Gly Arg

500 505 510

Ser Val Val Pro Ala Leu Leu Pro Leu Leu Ala Gly Thr Leu Leu Leu

515 520 525

Leu Glu Thr Ala Thr Ala Pro

530 535

<210> 3

<211> 1599

<212> DNA

<213> Homo sapiens

<400> 3

atgcaggggc cctgggtgct gctcctgctg ggcctgaggc tacagctctc cctgggcatc 60

atcccagttg aggaggagaa cccggacttc tggaaccgcc aggcagccga ggccctgggt 120

gccgccaaga agctgcagcc tgcacagaca gccgccaaga acctcatcat cttcctgggt 180

gacgggatgg gggtgtctac ggtgacagct gccaggatcc taaaagggca gaagaaggac 240

aaactggggc ctgagacctt cctggccatg gaccgcttcc cgtacgtggc tctgtccaag 300

acatacagtg tagacaagca tgtgccagac agtggagcca cagccacggc ctacctgtgc 360

ggggtcaagg gcaacttcca gaccattggc ttgagtgcag ccgcccgctt taaccagtgc 420

aacacgacac gcggcaacga ggtcatctcc gtgatgaatc gggccaagaa agcaggaaag 480

tcagtggggag tggtaaccac cacacgggtg cagcatgcct cgccagccgg cgcctacgcc 540

cacacggtga accgcaactg gtactcggat gccgacgtgc ctgcctcggc ccgccaggag 600

gggtgccagg acatcgccac gcagctcatc tccaacatgg acattgatgt gatcctaggt 660

ggaggccgaa agtacatgtt tcccatgggg accccagacc ctgagtaccc agatgactac 720

agccaaggtg ggaccaggct ggacgggaag aatctggtgc aggaatggct ggcgaagcac 780

cagggtgccc ggtacgtgtg gaaccgcact gagctcctgc aggcttccct ggacccgtct 840

gtgacccatc tcatgggtct ctttgagcct ggagacatga aatacgagat ccaccgagac 900

tccacactgg acccctccct gatggagatg acagaggctg ccctgctcct gctgagcagg 960

aacccccgcg gcttcttcct cttcgtggag ggtggtcgca tcgaccatgg tcatcatgaa 1020

agcagggctt accgggcact gactgagacg atcatgttcg acgacgccat tgagagggcg 1080

ggccagctca ccagcgagga ggacacgctg agcctcgtca ctgccgacca ctcccacgtc 1140

ttctccttcg gaggctaccc cctgcgaggg agctccatct tcgggctggc ccctggcaag 1200

gcccgggaca ggaaggccta cacggtcctc ctatacggaa acggtccagg ctatgtgctc 1260

aaggacggcg cccggccgga tgttacggag agcgagagcg ggagccccga gtatcggcag 1320

cagtcagcag tgcccctgga cggagagacc cacgcaggcg aggacgtggc ggtgttcgcg 1380

cgcggcccgc aggcgcacct ggttcacggc gtgcaggagc agaccttcat agcgcacgtc 1440

atggccttcg ccgcctgcct ggagccctac accgcctgcg acctggcgcc ccgcgccggc 1500

accaccgacg ccgcgcaccc ggggccgtcc gtggtccccg cgttgcttcc tctgctggca 1560

gggaccttgc tgctgctggg gacggccact gctccctga 1599

<210> 4

<211> 532

<212> PRT

<213> Homo sapiens

<400> 4

Met Gln Gly Pro Trp Val Leu Leu Leu Leu Gly Leu Arg Leu Gln Leu

1 5 10 15

Ser Leu Gly Ile Ile Pro Val Glu Glu Glu Asn Pro Asp Phe Trp Asn

20 25 30

Arg Gln Ala Ala Glu Ala Leu Gly Ala Ala Lys Lys Leu Gln Pro Ala

35 40 45

Gln Thr Ala Ala Lys Asn Leu Ile Ile Phe Leu Gly Asp Gly Met Gly

50 55 60

Val Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Lys Lys Asp

65 70 75 80

Lys Leu Gly Pro Glu Thr Phe Leu Ala Met Asp Arg Phe Pro Tyr Val

85 90 95

Ala Leu Ser Lys Thr Tyr Ser Val Asp Lys His Val Pro Asp Ser Gly

100 105 110

Ala Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Gly Asn Phe Gln Thr

115 120 125

Ile Gly Leu Ser Ala Ala Ala Arg Phe Asn Gln Cys Asn Thr Thr Arg

130 135 140

Gly Asn Glu Val Ile Ser Val Met Asn Arg Ala Lys Lys Ala Gly Lys

145 150 155 160

Ser Val Gly Val Val Thr Thr Thr Arg Val Gln His Ala Ser Pro Ala

165 170 175

Gly Ala Tyr Ala His Thr Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp

180 185 190

Val Pro Ala Ser Ala Arg Gln Glu Gly Cys Gln Asp Ile Ala Thr Gln

195 200 205

Leu Ile Ser Asn Met Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Lys

210 215 220

Tyr Met Phe Pro Met Gly Thr Pro Asp Pro Glu Tyr Pro Asp Asp Tyr

225 230 235 240

Ser Gln Gly Gly Thr Arg Leu Asp Gly Lys Asn Leu Val Gln Glu Trp

245 250 255

Leu Ala Lys His Gln Gly Ala Arg Tyr Val Trp Asn Arg Thr Glu Leu

260 265 270

Leu Gln Ala Ser Leu Asp Pro Ser Val Thr His Leu Met Gly Leu Phe

275 280 285

Glu Pro Gly Asp Met Lys Tyr Glu Ile His Arg Asp Ser Thr Leu Asp

290 295 300

Pro Ser Leu Met Glu Met Thr Glu Ala Ala Leu Leu Leu Leu Ser Arg

305 310 315 320

Asn Pro Arg Gly Phe Phe Leu Phe Val Glu Gly Gly Arg Ile Asp His

325 330 335

Gly His His Glu Ser Arg Ala Tyr Arg Ala Leu Thr Glu Thr Ile Met

340 345 350

Phe Asp Asp Ala Ile Glu Arg Ala Gly Gln Leu Thr Ser Glu Glu Asp

355 360 365

Thr Leu Ser Leu Val Thr Ala Asp His Ser His Val Phe Ser Phe Gly

370 375 380

Gly Tyr Pro Leu Arg Gly Ser Ser Ile Phe Gly Leu Ala Pro Gly Lys

385 390 395 400

Ala Arg Asp Arg Lys Ala Tyr Thr Val Leu Leu Tyr Gly Asn Gly Pro

405 410 415

Gly Tyr Val Leu Lys Asp Gly Ala Arg Pro Asp Val Thr Glu Ser Glu

420 425 430

Ser Gly Ser Pro Glu Tyr Arg Gln Gln Ser Ala Val Pro Leu Asp Gly

435 440 445

Glu Thr His Ala Gly Glu Asp Val Ala Val Phe Ala Arg Gly Pro Gln

450 455 460

Ala His Leu Val His Gly Val Gln Glu Gln Thr Phe Ile Ala His Val

465 470 475 480

Met Ala Phe Ala Ala Cys Leu Glu Pro Tyr Thr Ala Cys Asp Leu Ala

485 490 495

Pro Arg Ala Gly Thr Thr Asp Ala Ala His Pro Gly Pro Ser Val Val

500 505 510

Pro Ala Leu Leu Pro Leu Leu Ala Gly Thr Leu Leu Leu Leu Gly Thr

515 520 525

Ala Thr Ala Pro

530

<210> 5

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> mu 12F3 vH

<400> 5

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Ala Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Ser Ala Thr Tyr

85 90 95

Tyr Cys Ala Arg Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 6

<211> 101

<212> PRT

<213> Artificial Sequence

<220>

<223> mu IGHV7-3.04 - closest mouse germline V gene

<400> 6

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Ala Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Ser Ala Thr Tyr

85 90 95

Tyr Cys Ala Arg Asp

100

<210> 7

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> huIGHV3-49.01/hIGHJ4.01

<400> 7

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr

20 25 30

Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Thr Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Ser Lys Ser Ile

65 70 75 80

Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 8

<211> 100

<212> PRT

<213> Artificial Sequence

<220>

<223> hu IGHV3-72.01

<400> 8

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp His

20 25 30

Tyr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Thr Arg Asn Lys Ala Asn Ser Tyr Thr Thr Glu Tyr Ala Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg

100

<210> 9

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 vHA

<400> 9

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr

20 25 30

Tyr Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Ser Lys Ser Ile

65 70 75 80

Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 10

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 vHB

<400> 10

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Ile

65 70 75 80

Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 11

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 vHC

<400> 11

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ala Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Ile

65 70 75 80

Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 12

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 vHD

<400> 12

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 13

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 vHE

<400> 13

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ala Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 14

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 vHF

<400> 14

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ala Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 15

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 vHG

<400> 15

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ala Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Thr Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 16

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 vHH

<400> 16

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ala Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Thr Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 17

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> mu 12F3 vL

<400> 17

Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln Tyr Lys Thr Gly Lys Gly Pro Arg Leu Leu Ile

35 40 45

His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 18

<211> 95

<212> PRT

<213> Artificial Sequence

<220>

<223> mu IGKV19-93.01 - closest mouse germline V gene

<400> 18

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile

35 40 45

His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Leu

85 90 95

<210> 19

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> huIGKV1-33.01/hIGKJ2.01

<400> 19

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 20

<211> 95

<212> PRT

<213> Artificial Sequence

<220>

<223> hu IGKV1D-43.01

<400> 20

Ala Ile Arg Met Thr Gln Ser Pro Phe Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Trp Ala Ser Gln Gly Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Ala Lys Ala Pro Lys Leu Phe Ile

35 40 45

Tyr Tyr Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro

85 90 95

<210> 21

<211> 95

<212> PRT

<213> Artificial Sequence

<220>

<223> hu IGKV1-16.01

<400> 21

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro

85 90 95

<210> 22

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 vL1

<400> 22

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 23

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 vL2

<400> 23

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 24

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 vL3

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ser Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 25

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 vLA

<400> 25

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 26

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 vLB

<400> 26

Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 27

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 vLC

<400> 27

Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 28

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 vLD

<400> 28

Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ser Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 29

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 vLE

<400> 29

Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Tyr Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 30

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 vLF

<400> 30

Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Leu Ala Trp Tyr Gln Tyr Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Tyr Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 31

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 vLG

<400> 31

Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Thr Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Tyr Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 32

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 vLH

<400> 32

Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln Tyr Lys Thr Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 33

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 vLI

<400> 33

Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Phe Gln Tyr Lys Thr Gly Lys Ala Pro Lys Leu Phe Ile

35 40 45

His Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 34

<211> 453

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 HA heavy chain

<400> 34

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr

20 25 30

Tyr Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Ser Lys Ser Ile

65 70 75 80

Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 35

<211> 453

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 HB heavy chain

<400> 35

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Ile

65 70 75 80

Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 36

<211> 453

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 HC heavy chain

<400> 36

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ala Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Ile

65 70 75 80

Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 37

<211> 453

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 HD heavy chain

<400> 37

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 38

<211> 453

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 HE heavy chain

<400> 38

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ala Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 39

<211> 453

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 HF heavy chain

<400> 39

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ala Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 40

<211> 453

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 HG heavy chain

<400> 40

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ala Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Thr Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 41

<211> 453

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 HH heavy chain

<400> 41

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ala Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Thr Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 42

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 L1 light chain

<400> 42

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 43

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 L2 light chain

<400> 43

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 44

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 L3 light chain

<400> 44

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ser Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 45

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 LA light chain

<400> 45

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 46

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 LB light chain

<400> 46

Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 47

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 LC light chain

<400> 47

Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 48

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 LD light chain

<400> 48

Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ser Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 49

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 LE light chain

<400> 49

Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Tyr Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 50

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 LF light chain

<400> 50

Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Leu Ala Trp Tyr Gln Tyr Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Tyr Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 51

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 LG light chain

<400> 51

Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Thr Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Tyr Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 52

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 LH light chain

<400> 52

Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln Tyr Lys Thr Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 53

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 LI light chain

<400> 53

Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Phe Gln Tyr Lys Thr Gly Lys Ala Pro Lys Leu Phe Ile

35 40 45

His Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 54

<211> 330

<212> PRT

<213> Artificial Sequence

<220>

<223> hIgG1 heavy chain constant domain

<400> 54

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 55

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> hIgG kappa light chain constant domain

<400> 55

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 56

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> HF, HG CDR 1, Kabat

<400> 56

Asp Tyr Tyr Met Ser

1 5

<210> 57

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> HG CDR 2, Kabat

<400> 57

Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Thr Ala Ser

1 5 10 15

Val Lys Gly

<210> 58

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> HF, HG CDR 3, Kabat

<400> 58

Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

1 5 10

<210> 59

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> HF CDR 2, Kabat

<400> 59

Leu Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 60

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> HF, HG CDR 1, IMGT

<400> 60

Gly Phe Thr Phe Thr Asp Tyr Tyr

1 5

<210> 61

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> HF, HG CDR 2, IMGT

<400> 61

Ile Arg Asn Lys Ala Thr Gly Tyr Thr Thr

1 5 10

<210> 62

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> HF, HG CDR 3, IMGT

<400> 62

Ala Arg Ala Ser Phe Tyr Tyr Asp Gly Lys Val Leu Ala Tyr

1 5 10

<210> 63

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> LF CDR 1, Kabat

<400> 63

Gln Ala Ser Gln Asp Ile Asn Lys Tyr Leu Ala

1 5 10

<210> 64

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> LF CDR 2, Kabat

<400> 64

Tyr Thr Ser Ser Leu Gln Ser

1 5

<210> 65

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> LD, LF CDR 3, Kabat

<400> 65

Leu Gln Tyr Asp Asn Leu Tyr Thr

1 5

<210> 66

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> LD CDR 1, Kabat

<400> 66

Gln Ala Ser Gln Asp Ile Asn Lys Tyr Ile Ala

1 5 10

<210> 67

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> LD CDR 2, Kabat

<400> 67

Tyr Thr Ser Ser Leu Gln Pro

1 5

<210> 68

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> LD, LF CDR 1, IMGT

<400> 68

Gln Asp Ile Asn Lys Tyr

1 5

<210> 69

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> LD, LF CDR 2, IMGT

<400> 69

Tyr Thr Ser

1

<210> 70

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> LD, LF CDR 3, IMGT

<400> 70

Leu Gln Tyr Asp Asn Leu Tyr Thr

1 5

<210> 71

<211> 369

<212> DNA

<213> Artificial Sequence

<220>

<223> h12F3 vHG nucleic acid sequence

<400> 71

gaggtgcagc tggtggagtc cggaggagga ctggtgcagc ccggtcgttc tttaaggctg 60

agctgcacag ccagcggctt caccttcacc gactactaca tgtcttgggt gaggcaagct 120

cccggtaagg gactggagtg gctggcttta attcgtaaca aggccaccgg ctacaccacc 180

gagtacaccg cctccgtgaa gggtcgtttc accatctctc gtgacaacag caagtccatt 240

ttatatttac agatgaactc tttaaagacc gaggacaccg ccgtgtacta ctgcgctcgt 300

gcctcctttt actacgacgg caaggtgctg gcctactggg gccaaggtac tttagtgacc 360

gtgtcctcc 369

<210> 72

<211> 319

<212> DNA

<213> Artificial Sequence

<220>

<223> h12F3 vLF nucleic acid sequence

<400> 72

gacacccaga tgacccagtc cccttcctct ttatccgctt ccgtgggaga tcgtgtgacc 60

atcacttgtc aagcttccca agatatcaac aagtacctgg cttggtacca gtacaagccc 120

ggcaaggccc ccaagctgct gatccactac acctcctctt tacagtccgg agtgccttct 180

cgtttctccg gctccggaag cggtcgtgac tacaccttca ccatctcctc tttacagccc 240

gaggacatcg ctacctacta ctgtttacag tacgacaatt tatacacctt cggccaaggt 300

accaagctgg agatcaagc 319

<210> 73

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 epitope--ALPP

<400> 73

Leu Asp Pro Ser Val Thr His Leu Met Gly Leu Phe Glu Pro Gly Asp

1 5 10 15

Met Lys Tyr Glu Ile His Arg Asp Ser Thr Leu Asp Pro Ser Leu Met

20 25 30

Glu Met Thr Glu Ala Ala Leu Arg Leu Leu Ser

35 40

<210> 74

<211> 43

<212> PRT

<213> Artificial Sequence

<220>

<223> h12F3 epitope-ALPPL2

<400> 74

Leu Asp Pro Ser Val Thr His Leu Met Gly Leu Phe Glu Pro Gly Asp

1 5 10 15

Met Lys Tyr Glu Ile His Arg Asp Ser Thr Leu Asp Pro Ser Leu Met

20 25 30

Glu Met Thr Glu Ala Ala Leu Leu Leu Leu Ser

35 40

Claims (55)

1. An antigen-binding protein or fragment thereof that binds ALPP and/or ALPPL2, said antigen-binding protein or fragment thereof comprising the following 6 CDRs: CDR-H1 comprising the amino acid sequence of SEQ ID NO:56 or SEQ ID NO:60; CDR-H2 comprising the amino acid sequence of SEQ ID NO:57 or SEQ ID NO:61; CDR-H3 comprising the amino acid sequence of SEQ ID NO:58 or SEQ ID NO:62; CDR-L1 comprising the amino acid sequence of SEQ ID NO:63 or SEQ ID NO:68; A CDR-L2 comprising the amino acid sequence of SEQ ID NO:64 or SEQ ID NO:69; and CDR-L3 comprising the amino acid sequence of SEQ ID NO:65 or SEQ ID NO:70; Wherein the CDR is determined by Kabat or IMGT. 2. The antigen-binding protein or fragment of claim 1, comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 56; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 57; :CDR-H3 of the amino acid sequence of SEQ ID NO:58; CDR-L1 of the amino acid sequence of SEQ ID NO:63; CDR-L2 of the amino acid sequence of SEQ ID NO:64; and CDR-L1 of the amino acid sequence of SEQ ID NO:65 -L3; wherein said CDRs are determined by Kabat. 3. The antigen-binding protein or fragment of claim 1, comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 60; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 61; comprising SEQ ID NO :CDR-H3 of the amino acid sequence of SEQ ID NO:62; CDR-L1 of the amino acid sequence of SEQ ID NO:68; CDR-L2 of the amino acid sequence of SEQ ID NO:69; and CDR-L1 of the amino acid sequence of SEQ ID NO:70 -L3; wherein said CDR is determined by IMGT. 4. The antigen-binding protein or fragment of any one of claims 1-3, which comprises VH and VL, wherein the VH has at least 80%, 85%, or 90% of the amino acid sequence of SEQ ID NO: 15 , 95% or 99% amino acid sequence identity, and wherein the VL has at least 80%, 85%, 90%, 95% or 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO:30. 5. The antigen-binding protein or fragment of any one of claims 1-4, which comprises VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 15. 6. The antigen-binding protein or fragment of any one of claims 1-4, which comprises VH and VL, wherein the VL comprises the amino acid sequence of SEQ ID NO:30. 7. The antigen-binding protein or fragment of any one of claims 1-6, which comprises VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 15, and the VL comprises SEQ ID NO: 30 amino acid sequences. 8. The antigen-binding protein or fragment of any one of claims 1-7, which comprises: a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 40. 9. The antigen-binding protein or fragment of any one of claims 1-7, comprising: a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 50. 10. The antigen-binding protein or fragment of any one of claims 1-9, comprising: an HC comprising the amino acid sequence of SEQ ID NO: 40, and comprising: an LC comprising the amino acid sequence of SEQ ID NO: 50 . 11. The antigen-binding protein or fragment of any one of claims 1-10, wherein the antigen-binding protein is a monoclonal antibody or fragment thereof. 12. The antigen-binding protein or fragment of any one of claims 1-11, wherein the antigen-binding protein is a humanized antibody or fragment thereof. 13. The antigen binding protein or fragment of any one of claims 1-12, wherein the fragment is selected from the group consisting of Fab, Fab', Fv, scFv or (Fab') ₂ fragments. 14. An antibody-drug conjugate comprising the antibody or antigen-binding fragment of any one of claims 1-13 conjugated to a cytotoxic agent or cytostatic agent. 15. The antibody-drug conjugate of claim 14, wherein the antibody or antigen-binding fragment is conjugated to the cytotoxic agent or cytostatic agent via a linker. 16. The antibody-drug conjugate of any one of claims 14-15, wherein the cytotoxic agent or cytostatic agent is monomethyl auristatin. 17. The antibody-drug conjugate of any one of claims 14-16, wherein the monomethyl auristatin is monomethyl auristatin E (MMAE). 18. The antibody-drug conjugate of claim 17, wherein the antibody or antigen-binding fragment thereof is conjugated to MMAE via an enzyme-cleavable linker unit. 19. The antibody-drug conjugate of claim 18, wherein the enzyme-cleavable linker unit comprises a Val-Cit linker. 20. The antibody-drug conjugate of claim 19, wherein the antibody or antigen-binding fragment thereof is conjugated to MMAE through a linker unit having the following formula: -A _a -W _w -Y _y -; wherein - A- is an extension unit, a is 0 or 1; -W- is an amino acid unit, w is an integer ranging from 0 to 12; and -Y- is a spacer unit, y is 0, 1 or 2. 21. The antibody-drug conjugate of claim 20, wherein the extension unit has the structure of Formula I; wherein the amino acid unit is Val-Cit; and wherein the spacer unit is of Formula II The p-aminobenzyl alcohol (PABC) group of the structure; 22. The antibody-drug conjugate of any one of claims 14-21, wherein the linker is connected to monomethyl auristatin E, thereby forming an antibody-drug conjugate having the following structure: where Ab is antibody h12F3 and p represents a number from 1 to 16. 23. The antibody-drug conjugate of claim 22, wherein the average value of p in the population of antibody-drug conjugates is about 4. 24. The antibody-drug conjugate of any one of claims 14-18, wherein the antibody-drug conjugate is represented by the following structure: or a pharmaceutically acceptable salt thereof, wherein: Ab is antibody h12F3 and p represents a number from 1 to 12; The subscript nn is a number from 1 to 5; The subscript a' is 0, and A' does not exist; P1, P2 and P3 are each an amino acid, where: The first one of the amino acids P1, P2 or P3 is negatively charged; The second of said amino acids P1, P2 or P3 has an aliphatic side chain that is no more hydrophobic than leucine; and The third of said amino acids P1, P2 or P3 is less hydrophobic than leucine, wherein said first of said amino acids P1, P2 or P3 corresponds to any one of P1, P2 or P3 and said second of said amino acids P1, P2 or P3 corresponds to the two remaining amino acids P1 , one of P2 or P3, and said third of said amino acids P1, P2 or P3 corresponds to the last remaining amino acid P1, P2 or P3, The condition is that -P3-P2-P1- is not -Glu-Val-Cit- or -Asp-Val-Cit-. 25. The antibody-drug conjugate of claim 24, wherein subscript nn is 2. 26. The antibody-drug conjugate of claim 24 or 25, wherein: The P3 amino acid of the tripeptide has a D-amino acid configuration; One of the P2 and P1 amino acids has an aliphatic side chain that is less hydrophobic than leucine; and The other one of the P2 and P1 amino acids is negatively charged. 27. The antibody-drug conjugate of any one of claims 24-26, wherein the P3 amino acid is D-Leu or D-Ala. 28. The antibody-drug conjugate of any one of claims 24-27, wherein the P3 amino acid is D-Leu or D-Ala, the P2 amino acid is Ala, Glu or Asp, and the The P1 amino acid is Ala, Glu or Asp. 29. The antibody-drug conjugate of any one of claims 24-28, wherein -P3-P2-P1- is -D-Leu-Ala-Asp-, -D-Leu-Ala-Glu- , -D-Ala-Ala-Asp- or -D-Ala-Ala-Glu-. 30. The antibody-drug conjugate of any one of claims 24-29, wherein -P3-P2-P1- is -D-Leu-Ala-Glu-. 31. The antibody-drug conjugate of any one of claims 24-30, wherein the antibody-drug conjugate is represented by the following structure: or a pharmaceutically acceptable salt thereof, where Ab is antibody h12F3 and p represents a number from 1 to 12. 32. An isolated nucleic acid encoding the antigen-binding protein or fragment of any one of claims 1-13. 33. A vector comprising the nucleic acid of claim 32. 34. A host cell comprising the vector of claim 33. 35. The host cell of claim 34, wherein the host cell is a CHO cell. 36. A host cell producing the antigen-binding protein or fragment of any one of claims 1-13. 37. A method of preparing an antigen-binding protein or a fragment thereof, the method comprising culturing the host cell of claim 36 under conditions suitable for producing the antigen-binding protein. 38. The method of claim 37, further comprising recovering the antigen-binding protein or fragment produced by the host cell. 39. The method of claim 37 or claim 38, wherein the host cell is a CHO cell. 40. An antigen-binding protein or fragment thereof produced by the method of any one of claims 37-39. 41. A pharmaceutical composition comprising the antigen-binding protein or fragment of any one of claims 1-31 or 40 and a pharmaceutically acceptable carrier. 42. A method of treating cancer expressing ALPP and/or ALPPL2 in an individual, comprising administering an effective amount of the antigen-binding protein or fragment of any one of claims 1-31 or 40 to an individual in need thereof, Or the pharmaceutical composition according to claim 41. 43. The method of claim 42, wherein the cancer is ovarian, lung, endometrial, bladder, gastric or testicular cancer. 44. The method of claim 43, wherein the cancer is ovarian cancer. 45. An antibody-drug conjugate comprising an isolated anti-ALPP/ALPPL2 antibody conjugated to mc-vc-PABC-MMAE, wherein the antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO: 15. A variable region and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30, and wherein the antibody-drug conjugate has the following structure: where Ab is the antibody and p represents a number from 1 to 16. 46. An antibody-drug conjugate comprising an antigen-binding protein that binds ALPP and ALPPL2 or a fragment thereof, wherein the antibody-drug conjugate is represented by the following structure: or a pharmaceutically acceptable salt thereof, wherein: Ab is anti-ALPP/ALPPL2 antibody and p represents a number from 1 to 12; The subscript nn is a number from 1 to 5; The subscript a' is 0, and A' does not exist; P1, P2 and P3 are each an amino acid, where: The first one of the amino acids P1, P2 or P3 is negatively charged; The second of said amino acids P1, P2 or P3 has an aliphatic side chain that is no more hydrophobic than leucine; and The third of said amino acids P1, P2 or P3 is less hydrophobic than leucine, wherein said first of said amino acids P1, P2 or P3 corresponds to any one of P1, P2 or P3 and said second of said amino acids P1, P2 or P3 corresponds to the two remaining amino acids P1 , one of P2 or P3, and said third of said amino acids P1, P2 or P3 corresponds to the last remaining amino acid P1, P2 or P3, The condition is that -P3-P2-P1- is not -Glu-Val-Cit- or -Asp-Val-Cit-. 47. The antibody-drug conjugate of claim 46, wherein subscript nn is 2. 48. The antibody-drug conjugate of claim 46 or 47, wherein: The P3 amino acid of the tripeptide has a D-amino acid configuration; One of the P2 and P1 amino acids has an aliphatic side chain that is less hydrophobic than leucine; and The other one of the P2 and P1 amino acids is negatively charged. 49. The antibody-drug conjugate of any one of claims 46-48, wherein the P3 amino acid is D-Leu or D-Ala. 50. The antibody-drug conjugate of any one of claims 46-49, wherein said P3 amino acid is D-Leu or D-Ala, said P2 amino acid is Ala, Glu or Asp, and said The P1 amino acid is Ala, Glu or Asp. 51. The antibody-drug conjugate of any one of claims 46-50, wherein -P3-P2-P1- is -D-Leu-Ala-Asp-, -D-Leu-Ala-Glu- , -D-Ala-Ala-Asp- or -D-Ala-Ala-Glu-. 52. The antibody-drug conjugate of any one of claims 46-51, wherein -P3-P2-P1- is -D-Leu-Ala-Glu-. 53. The antibody-drug conjugate of any one of claims 46-52, wherein the antibody-drug conjugate is represented by the following structure: or a pharmaceutically acceptable salt thereof, where Ab is an anti-ALPP/ALPPL2 antibody and p represents a number from 1 to 12. 54. An antibody-drug conjugate comprising an isolated anti-ALPP/ALPPL2 antibody conjugated to mp-dLAE-PABC-MMAE, wherein the antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO: 15. A variable region and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30, and wherein the antibody-drug conjugate has the following structure: where Ab is the antibody and p represents a number from 1 to 12. 55. An antigen-binding protein or fragment thereof that binds ALPP and/or ALPPL2 and is capable of binding one or more amino acids of a peptide comprising SEQ ID NO:73 and/or SEQ ID NO:74 .