CN116997356A - anti-ALPP/ALPPL 2 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 making such antigen binding proteins are also provided. The antigen binding proteins are useful in a variety of methods, including the treatment of ovarian cancer.

Description

anti-ALPP/ALPPL 2 antibodies and antibody-drug conjugates

Cross Reference to Related Applications

The application claims the benefit of U.S. provisional application Ser. No. 63/162,635, filed on 18 at 3 months of 2021, and U.S. provisional application Ser. No. 63/301,574, filed on 21 at 1 months of 2022, each of which is incorporated by reference in its entirety for all purposes.

Reference to sequence Listing

The present application includes an electronic sequence listing in a file created by 2022, 3, 4, entitled 5620-00112pc_st25 and containing 106Kb, which is hereby incorporated by reference.

Technical Field

The present application relates to novel anti-ALPP/ALPPL 2 antibodies and antibody-drug conjugates and methods of treating cancer using such anti-ALPP/ALPPL 2 antibodies and antibody-drug conjugates.

Background

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

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

Disclosure of Invention

Provided herein are anti-ALPP antibodies and antibody-drug conjugates (ADCs) to ALPP, and anti-ALPPL 2 antibodies and ADCs to ALPPL 2. Also provided herein are antibodies that can bind both ALPP and ALPPL2 (anti-ALPP/ALPPL 2 antibodies) and ADCs for both ALPP and ALPPL2 (ADCs for ALPP/ALPPL 2). Also provided herein are methods of treating ALPP and/or ALPPL2 expressing disorders (including cancer) using antibodies and ADCs directed against ALPP/ALPPL 2. In some embodiments, the anti-ALPP/ALPPL 2 antibody comprises the heavy chain CDR sequences of SEQ ID NOS 56, 57 and 58 and the light chain CDR sequences of SEQ ID NOS 63, 64 and 65, as determined by the Kabat numbering. In some embodiments, the anti-ALPP/ALPPL 2 antibody comprises the heavy chain CDR sequences of SEQ ID NOS 60, 61 and 62 and the light chain CDR sequences of SEQ ID NOS 66, 67 and 68, as determined by IMGT numbering.

Drawings

FIG. 1 shows cytotoxicity assays for ALPP/ALPPL2 specific antibodies as ADCs using payloads with and without bystander activity against COV644 and NCI-H1651 tumor cells.

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

FIG. 3 shows the binding affinity of ALPP/ALPPL2 specific antibodies.

Figure 4 shows the complete binding profile of antibodies 1F7 and 12F3 to HEK293 cells expressing ALPP and ALPPL2 as measured by flow cytometry.

FIG. 5 shows a sequence alignment of the h12F3 variable heavy chain variant with the human heavy chain acceptor sequence IGHV3-49/HJ 4.

FIG. 6 shows a variable domain alignment of h12F3 variable heavy chain variants.

FIG. 7 shows sequence alignment of h12F3 variable light chain variants with human kappa receptor sequence IGKV1-33/KJ 2.

FIG. 8 shows a variable domain alignment of h12F3 light chain variants.

Figure 9 shows the percentage of surviving CAOV3 cells. The left side of the figure shows the percent viability of the dose-response curves for ADCs containing humanized F light chain variants and different heavy chains. The right side of the figure shows the viability of ADCs containing humanized D heavy and different light chain variants.

Figure 10 shows in vitro potency correlation between humanized variants with mp-dLAE-MMAE (4) drug linkers.

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 to ALPP and ALPPL2 Fc fusions are shown.

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

Figure 13 shows the kinetic binding curves and values of h12F3 HGLF and HFLD variants to ALPP and ALPPL2 Fc fusions.

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

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

FIG. 16 shows the in vivo antitumor activity of h12F3 HGLF-dLAE-MMAE and HFLD-dLAE-MMAE in NCI-N87 mouse models.

FIG. 17 shows the in vivo antitumor activity of h12F3 HFLD-dLAE-MMAE and HFLD-vc-MMAE in NCI-N87 mouse models.

FIG. 18 shows the in vivo antitumor activity of H12F3 HFLD-dLAE-MMAE and HFLD-vc-MMAE in the H1651 mouse model.

Figure 19 shows% change in tumor volume in seven xenograft models after treatment with h12F3ADC conjugated to vc-MMAE and dLAE-MMAE.

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

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

FIG. 22 shows a summary of tumor growth inhibition by h12F3-HGLF mc-vc-MMAE and mp-dLAE-MMAE for four xenograft tumor models including pancreas (HPAC), stomach (NCI-N87), ovary (CAOV 3) and lung (SNU-2535). The average non-target ADC is shown as a dashed line.

FIG. 23 shows the antitumor activity of ovarian patient-derived xenografts treated with h12F 3-HDLF-mc-vc-MMAE. A) shows a summary of tumor growth inhibition for twelve xenografts, while B) and C) show examples of two conjugate-treated models compared to untreated cohorts.

Fig. 24 shows the binding affinities of h12F3 HGLF and HFLD for human ALPP and monkey ortholog ALPP.

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

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

FIG. 27 shows cell lysis of LoVo cells as determined by a chromium release assay after incubation of Na2[51Cr ] O4 (Cr-51) labeled cells with NK cells in the presence of h12F3HGLF, h12F3HGLF-mc-vc-MMAE or h12F3 HGLF-mp-dLAE-MMAE conjugates.

FIG. 28 shows phagocytic activity of macrophages incubated with LoVo cells in the presence of h12F3HGLF, h12F3HGLF-mc-vc-MMAE or h12F3 HGLF-mp-dLAE-MMAE conjugates compared to positive (function blocking anti-CD 47 antibodies) or isotype controls.

FIG. 29 shows FcgRIII mediated activation of the luminescent reporter by the h12F3HGLF antibody and ADC.

FIG. 30 shows the activation of two signaling pathways involved in immunogenic cell death by two different concentrations of h12F3HGLF-mc-vc-MMAE or h12F3 HGLF-mp-dLAE-MMAE conjugates.

FIG. 31 shows ATP release in culture medium of LoVo cells treated with 1 or 10mg/ml h12F3HGLF-mc-vc-MMAE or h12F3 HGLF-mp-dLAE-MMAE conjugate 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 cited herein are generally well understood and commonly used by those skilled in the art using conventional methods, such as the widely used methods described in the following documents: sambrook et al Molecular Cloning: A Laboratory Manual, 4 th edition (2012) Cold Spring Harbor Laboratory Press, cold Spring Harbor n.y.; current Protocols In Molecular Biology (F.M. Ausubel et al, (2003)); METHODS IN ENZYMOLOGY series (Academic Press, inc.); PCR 2:A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor editions (1995)); greenfield editions (2013) Antibodies, A Laboratory Manual, 2 nd edition, cold Spring Harbor Laboratory Press; oligonucleotide Synthesis (m.j. Gait edit, 1984); methods in Molecular Biology, humana Press; cell Biology A Laboratory Notebook (J.E.Cellis editions, 1998) Academic Press; animal Cell Culture (r.i. freshney) edit, 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 eds., 1993-8) J. Wiley and Sons; handbook of Experimental Immunology (d.m. weir and c.c. blackwell editions); gene Transfer Vectors for Mammalian Cells (J.M.Miller and M.P.Calos. Editions, 1987); PCR: the Polymerase Chain Reaction, (Mullis et al, 1994); current Protocols in Immunology (J.E. Coligan et al, editions, 1991); short Protocols in Molecular Biology (Wiley and Sons, 1999); immunobiology (c.a. janeway and p.convers, 1997); antibodies (P.Finch, 1997); antibodies A Practical Approach (D.Catty. Eds., IRL Press, 1988-1989); monoclonal Antibodies: A Practical Approach (P.shepherd and C.dean editions, 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 editions Harwood Academic Publishers, 1995), cancer Principles and Practice of Oncology (V.T.DeVita et al editions, J.B.Lippincott Company, 1993), and updated versions thereof, each of The foregoing references in this paragraph are incorporated by reference in their entirety.

I. Definition of the definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, concise Dictionary of Biomedicine and Molecular Biology, juo, pei-Show, 2 nd edition, 2002, CRC Press; the Dictionary of Cell and Molecular Biology, 5 th edition, 2013,Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, 2 nd edition, 2006,Oxford University Press, provide the skilled artisan with a general dictionary of many terms used in the present disclosure.

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

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

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

As used herein, the term "and/or" should be taken as a specific disclosure of each of two particular 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 a phrase such as "A, B and/or C" is intended to encompass each of the following aspects: 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); c (alone).

The term "about" refers to a value or composition that is within acceptable error limits for the 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 understood by those of skill in the art, references herein to "about" a value or parameter include (and describe) embodiments directed to 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 fractions thereof (such as tenths and hundredths of integers) as appropriate, unless otherwise indicated.

When trade names are used herein, reference to trade names also refers to product formulations, imitation pharmaceuticals, and active pharmaceutical ingredients of trade name products, 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 variant (e.g., splice variant, allelic variant), allotype, and vertebrate species homolog of human ALPP. The term encompasses "full length", unprocessed ALPP, and any form of ALPP produced by intracellular processing. Uniprot ID: p05187 or RefSeq ID: the amino acid sequence of an exemplary human ALPP is provided in nm_ 001632. The amino acid sequence of a specific example of mature human ALPP protein is shown in SEQ ID NO. 2.

The terms "ALPPL2," "placenta-like alkaline phosphatase 2," or "germ cell alkaline phosphatase" are used interchangeably herein and include, unless otherwise indicated, any naturally occurring variant (e.g., splice variant, allelic variant), allotype, and vertebrate species homolog of human ALPPL2. The term encompasses "full length", unprocessed ALPPL2, and any form of ALPPL2 produced by intracellular processing. Uniprot ID: p10696 or RefSeq ID: an exemplary amino acid sequence of human ALPPL2 is provided in nm_ 031313. A specific example of an mature human ALPPL2 protein has the amino acid sequence shown in SEQ ID NO. 4.

As used herein, "antigen binding protein" ("ABP") refers to any protein that binds a particular target antigen, and not to a naturally occurring cognate ligand or fragment of such a ligand that binds the particular antigen. In the present application, the specific target antigen is ALPP and/or ALPPL2 or a fragment of ALPP and/or ALPPL2. An "antigen binding protein" includes a protein comprising 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 linked. In some antigen binding proteins, the HVRs or CDRs are embedded into a "framework" region that orients the HVRs or CDRs such that the appropriate antigen binding properties of the CDRs are achieved. For some antigen binding proteins, the scaffold is an immunoglobulin heavy 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 10 th extracellular domain of human fibronectin III), neomycin CBM4-2, lipocalin-derived anticalin, engineered ankyrin repeat domain (DARPin), protein-a domain (protein Z), kunitz domain, im9, TPR protein, zinc finger domain, pVIII, GC4, transferrin, B domain of SPA, sac7d, a-domain, SH3 domain of Fyn kinase, and C-type lectin-like domain (see, e.g., gebauer and Skerra (2009) curr. Chem. Biol.,13:245-255; binz et al (2005) na. 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 mimics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions, and portions or fragments of each. In some casesThe antigen binding proteins are functional fragments of whole antibodies (e.g., fab ', F (ab') ₂ scFv, domain antibody or minibody). A peptibody is another example of an antigen binding protein. In some embodiments, the term "antigen binding protein" includes derivatives, e.g., antigen binding proteins that have been chemically modified, e.g., 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), which retains the ability to specifically bind to an antigen bound by the entire antigen binding protein, regardless of how obtained or synthesized. Examples of antibody fragments include, but are not limited to, fv; fab; fab'; fab' -SH; f (ab') ₂ The method comprises the steps of carrying out a first treatment on the surface of the A diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. An "Fv" fragment comprises a non-covalently linked dimer of one heavy chain variable domain and one light chain variable domain. "Fab" fragments include, 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 of the heavy chain (C _H1 )。“F(ab') ₂ "fragments include two Fab fragments linked by a disulfide bond near the hinge region.

The terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues and are not limited to a minimum length. Polymers of such amino acid residues may contain natural or unnatural amino acid residues, and include, but are not limited to, dimers, trimers, peptides, oligopeptides, and multimers 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" is also intended to include modifications to the native sequence, such as deletions, additions and substitutions (which are generally 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 with 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 naturally occurring polypeptide. Thus, a native sequence polypeptide may have the amino acid sequence of a naturally occurring polypeptide from any mammal. Such native sequence polypeptides may be isolated from nature or may be produced by recombinant or synthetic means. The term "native sequence" polypeptide specifically encompasses naturally occurring truncated or secreted forms (e.g., extracellular domain sequences) of the polypeptide, naturally occurring variant forms (e.g., alternatively spliced forms) and naturally occurring allelic variants of the polypeptide.

By polypeptide "variant" is meant a biologically active polypeptide (e.g., an antigen binding protein or antibody) that has at least about 70%, 80%, or 90% amino acid sequence identity to a native or reference sequence polypeptide after aligning the sequences and introducing gaps (if desired) to achieve the maximum percent sequence identity and without regard to 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 to the 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 is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity and not taking any conservative substitutions into account as part of the sequence identity. For determining the percentage of amino acid sequencesThe alignment for identity can be accomplished in a variety of ways within the skill of the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN ^TM (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences compared. For example, the sequence identity (which may alternatively be expressed as a given amino acid sequence a having or comprising a certain% of sequence identity to, with or against a given amino acid sequence B) of a given amino acid sequence a pair, with or against a given amino acid sequence B is calculated as follows:

100 times the fraction X/Y

Wherein X is the number of amino acid residues scored as identical matches by the sequences in the alignment of programs a and B, and wherein Y is the total number of amino acid residues in B. All% amino acid sequence identity values used herein are calculated according to this formula using the ALIGN-2 computer program unless specifically stated otherwise. It will be appreciated 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 facilitates secretion of the polypeptide from a mammalian cell. The leader sequence may be cleaved upon export of the polypeptide from the mammalian cell to form the mature protein. The leader sequences may be natural or synthetic and they may be heterologous or homologous to the protein to which they are attached.

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 chains being interconnected by disulfide bonds. The structure of immunoglobulins is well characterized. See, e.g., fundamental Immunology (Paul, W.edit., 7 th edition Raven Press, N.Y. (2013)). In brief, each heavy chain typically comprises a heavy chain variable region (abbreviated herein as V _H Or VH) and a heavy chain constant region (C _H Or CH). Heavy chain constant regions typically comprise three domains: c (C) _H 1、C _H 2 andC _H 3. heavy chains are typically linked to each other by disulfide bonds in the so-called "hinge region". Each light chain typically comprises a light chain variable region (abbreviated herein as V _L Or VL) and a light chain constant region (C _L Or CL). The light chain constant region generally comprises a domain C _L . CL may be the kappa (kappa) or lambda (lambda) isoform. The terms "constant domain" and "constant region" are used interchangeably herein. The immunoglobulin may be derived from any generally known isotype, including but not limited to IgA, secretory IgA, igG, and IgM. Subclasses of IgG 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 class or subclass of antibodies (e.g., igM or IgG 1) encoded by the heavy chain constant region gene.

The term "antibody" is used in its broadest sense and specifically covers, for example, monoclonal antibodies (including full length or intact monoclonal antibodies), antibodies with multi-or mono-epitope specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), single chain antibodies, and fragments of the foregoing 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, and the like. Thus, the term "antibody" includes polypeptide products such as B cells within an immunoglobulin-like polypeptide that are capable of binding a particular molecular antigen and that 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 comprises a variable region of about 100 to about 130 amino acids or more, and each carboxy-terminal portion of each chain comprises a constant region. See, for example Antibody Engineering(Borrebaeck edition, 2 nd edition, 1995); the number of bits of the block in the block is Kuby,Immunology(3 rd edition, 1997). The term "antibody" also includes, but is not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intracellular antibodies, anti-idiotype (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the foregoing antibodies, which refer to a portion of an antibody heavy and/or light chain polypeptide that retains the antibody from which the fragment was derivedSome or all of the binding activity. 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, F (ab') ₂ Fragments, disulfide-linked Fv (dsFv), 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 that contain an antigen binding site (e.g., one or more CDRs of an antibody) that binds an antigen. Such antibody fragments can be found in, for example, harlow and Lane,Antibodies:A Laboratory Manual(1989)；Mol.Biology and Biotechnology:A ComprehensiveDesk Reference(Myers editions, 1995); huston et al, 1993,Cell Biophysics 22:189-224; pluckthun and Skerra,1989, meth. Enzymol.178:497-515; and (c) a step of obtaining a product, Advanced Immunochemistry(2 nd edition, 1990). The immunoglobulins disclosed herein can be of any type (e.g., igG, igE, igM, igD and IgA) or subclass (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2) of immunoglobulin molecule.

As used herein, the term "hypervariable region" or "HVR" refers to each region of sequence hypervariability in an antibody variable domain. The HVR can form a structurally defined ring ("hypervariable ring"). Typically, a natural four-chain antibody comprises six HVRs; three of the VH (H1, H2, H3) and three of the VL (L1, L2, L3). Of the natural antibodies, H3 and L3 show the greatest diversity among the six HVRs, and in particular H3 is believed to play a unique role in conferring good specificity to the antibody. See, e.g., xu et al, immunity 13:37-45 (2000); johnson and Wu, methods in Molecular Biology 248:1-25 (Lo editor, human Press, totowa, N.J., 2003). In fact, naturally occurring camelid antibodies consisting of heavy chains only are functional and stable in the absence of light chains. See, e.g., hamers-Casterman et al, nature 363:446-448 (1993); sheiff et al Nature Structure. Biol.3:733-736 (1996).

HVRs typically comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs) that have the highest sequence variability and/or are involved in antigen recognition. Various schemes for defining boundaries for a given CDR are known in the art. For example, kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are most commonly used (Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, MD. (1991)). Chothia instead refers to the position of the structural ring (Chothia and Lesk, J.mol. Biol.196:901-917 (1987)). The AbM CDRs represent a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular AbM antibody modeling software. The "contact" CDR is based on analysis of available complex crystal structures. Additional details regarding the foregoing schemes 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 honeygger a. And plurkthun a. (2001) j.mol/biol.309:657-70, (AHo numbering scheme).

In some embodiments, the HVR regions and related sequences are identical to CDR regions and related sequences based on one of the numbering conventions described above. Thus, the residues of exemplary HVRs and/or CDRs are summarized in table 1 below.

Table 1: summary of different CDR numbering schemes

Ring(s)	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 respective CDRs (e.g., CDR-H1, CDR-H2) of the antibody or region thereof are to be understood as encompassing the complementarity determining regions as defined by any known scheme as described above. In some cases, a scheme for identifying one or more particular CDRs is specified, such as CDRs defined by 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 proteins comprise HVR and/or CDR residues as identified in fig. 5-8 or as set forth elsewhere herein.

The term "variable region" or "variable domain" refers to a domain of an antigen binding protein (e.g., an antibody) heavy or light chain that is involved in binding of the antigen binding protein (e.g., an antibody) to an antigen. The variable regions or domains of antigen binding proteins such as the heavy and light chains (VH and VL, respectively) of antibodies may be further subdivided into regions of hypervariability (or hypervariable regions, which may be hypervariable in the form of sequence and/or structurally defined loops), such as hypervariable regions (HVRs) or Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, referred to as Framework Regions (FR). 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 regions" and "FR" are known in the art and refer to the non-HVR or non-CDR portions 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 carboxy-terminus in the following order: FR1, HVR1, FR2, HVR2, FR3, HVR3, FR4 in the case of HVR, 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. Alternatively, VH or VL domains from antigen-binding antibodies may be used to isolate antibodies that bind to a particular antigen to screen libraries of complementary VL or VH domains, respectively. See, for example, 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 chains HVR-H1, FR-H2, HVR-H2, FR-H3 and HVR-H3. For example, the heavy chain variable region may comprise heavy chains 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.

The term "heavy chain constant region" as used herein refers to a heavy chain comprising at least three heavy chain constant domains C _H 1、C _H 2 and C _H 3. Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha. Non-limiting exemplary heavy chainsThe constant region also includes epsilon and mu. Each heavy chain constant region corresponds to an antibody isotype. For example, the antibody comprising a gamma constant region is an IgG antibody, the antibody comprising a delta constant region is an IgD antibody, and the antibody comprising an alpha constant region is an IgA antibody. Furthermore, the antibody comprising the μ constant region is an IgM antibody, and the antibody comprising the ε constant region is an IgE antibody. Certain isoforms may be further subdivided into subclasses. For example, igG antibodies include, but are not limited to, igG1 (including gamma ₁ Constant region), igG2 (comprising gamma ₂ Constant region), igG3 (comprising gamma ₃ Constant region) and IgG4 (comprising gamma ₄ Constant region) antibodies; igA antibodies include, but are not limited to IgA1 (comprising alpha ₁ Constant region) and IgA2 (comprising alpha ₂ 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 chains HVR-L1, FR-L2, HVR-L2, FR-L3 and HVR-L3. In some embodiments, the light chain variable region comprises light chains 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.

The term "light chain constant region" as used herein refers to a region comprising a light chain constant domain C _L Is a region of (a) in the above-mentioned region(s). Non-limiting exemplary light chain constant regions include lambda and kappa.

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 immunoglobulin heavy chain constant region, 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, 5 th edition, 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 numbering in the constant domains of antibodies refer to residue numbering by the EU numbering system.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific for a single antigenic site. In contrast to polyclonal antibody preparations, which may include different antibodies directed against 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 epitopes. In one embodiment, the epitopes are from the same antigen. In another embodiment, the epitope is from two different antigens. Methods for preparing bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly using co-expression of two immunoglobulin heavy chain/light chain pairs. See, e.g., milstein et al, nature305:537-39 (1983). Alternatively, bispecific antibodies can be prepared using chemical ligation. See, e.g., brennan et al, science 229:81 (1985). Bispecific antibodies include bispecific antibody fragments. See, e.g., 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 immunoglobulins" or "DVD-Ig" refer to multivalent and multispecific binding proteins as described, for example, 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 forms of bispecific antibodies in which the heavy variable domain of one antibody is linked to the light variable domain of the other antibody and the two chains are associated and are described, for example, in Garber, nature Reviews Drug Discovery13:799-801, 2014.

"bispecific T cell engagers" orIs a genetic fusion of two scFv fragments that produce tandem scFv molecules 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 and/or light chains are derived from a particular source or species, while the remainder of the heavy and/or light chains are derived from a different source or species. In some embodiments, chimeric antibodies refer to antibodies comprising at least one variable region from a first species (such as mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (such as human, cynomolgus monkey, etc.). In some embodiments, the chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, the chimeric antibody comprises at least one cynomolgus monkey variable region and at least one human constant region. In some embodiments, all variable regions of the chimeric antibody are from a first species and all constant regions of the chimeric antibody are from a second species.

As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody that comprises human antibody constant domains and non-human variable domains modified to comprise a high level of sequence homology to human variable domains. This can be achieved by grafting six non-human antibody Complementarity Determining Regions (CDRs) onto a cognate human acceptor Framework Region (FR) (see WO92/22653 and EP 0629240). In order to fully reconstruct the binding affinity and specificity of a parent antibody, it may be desirable to replace the framework residues from the parent antibody (i.e., the non-human antibody) with human framework regions (back mutations). Structural homology modeling can help identify amino acid residues in the framework regions that are important for the binding properties of antibodies. Thus, a humanized antibody may comprise non-human CDR sequences, predominantly human framework regions, optionally comprising one or more amino acid back mutations to the non-human amino acid sequence; a fully human constant region. Optionally, additional amino acid modifications, not necessarily back mutations, may be applied to obtain humanized antibodies with preferred characteristics such as affinity and biochemical properties.

As used herein, "human antibody" refers to an antibody produced in a human, an antibody produced in a non-human animal comprising human immunoglobulin genes, such as And antibodies selected using ex vivo methods such as phage display, wherein the antibody repertoire is based on human immunoglobulin sequences. A "human antibody" is an antibody having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The 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 mutation 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 grafted onto human framework sequences. The terms "human antibody" and "fully human antibody" are used synonymously.

A "recipient human framework" for purposes herein is a framework comprising an amino acid sequence derived from a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework of a human immunoglobulin framework or a human consensus framework as defined below. The acceptor human framework derived from the human immunoglobulin framework or the human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence variations. 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 the human consensus framework sequence.

An "affinity matured" antibody refers to an antibody having one or more alterations in one or more hypervariable regions (HVRs) that result in an improvement in the affinity of the antibody for an antigen as compared to the parent antibody that does not have such alterations. In some examples, affinity matured antibodies refer to antibodies having one or more alterations in one or more Complementarity Determining Regions (CDRs) that result in an improvement in the affinity of the antibody for the antigen as compared to the parent antibody that does not have such alterations.

The term "derivative" refers to a molecule (e.g., an antigen binding protein such as an antibody or fragment thereof) that includes chemical modifications other than insertions, deletions, or substitutions of amino acids (or nucleic acids). In certain embodiments, the derivatives comprise covalent modifications, including, but not limited to, chemical bonding to polymers, lipids, or other organic or inorganic moieties. In certain embodiments, derivatives of a particular antigen binding protein may have a longer circulatory half-life than an antigen binding protein that has not been chemically modified. In certain embodiments, the derivatives may have improved targeting ability to a desired cell, tissue and/or organ. In some embodiments, derivatives of antigen binding proteins are 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-vinylpyrrolidone) -polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), and polyvinyl alcohol, as well as mixtures of such polymers. See, for example, U.S. Pat. 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 ALPPL 2) to which an antigen binding protein (e.g., antibody or fragment thereof) that targets the antigen binds. Epitopes generally consist of chemically active surface groups of molecules such as amino acids, polypeptides, sugar side chains, phosphoryl groups or sulfonyl groups, and have specific three-dimensional structural features as well as specific charge characteristics. Epitopes can be formed by contiguous or non-contiguous amino acids of antigens juxtaposed by tertiary folding. Epitopes formed by consecutive residues are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment 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, 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 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 length. In one embodiment, the epitope of the anti-ALPP/ALPPL 2 antibody of the invention comprises SEQ ID NO:73 and/or SEQ ID NO:74. An epitope may comprise amino acid residues that are directly involved in binding (also referred to as immunodominant components 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 an antigen binding molecule (i.e., amino acids within the footprint of the antigen binding molecule). Methods of 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, volume 66, edited by g.e.morris (1996)). Once the desired epitope of an antigen is determined, established techniques can be used to generate antigen binding proteins (e.g., antibodies or fragments thereof) directed against the epitope. 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 "nonlinear epitope" or "conformational epitope" comprises a discontinuous polypeptide, amino acid and/or sugar within an antigenic protein to which an antibody specific for the epitope binds.

A "linear epitope" comprises a contiguous polypeptide, amino acid, and/or sugar within an antigen protein to which an antigen binding protein (e.g., an antibody or fragment thereof) specific for that epitope binds.

The term "compete" when used in the context of antigen binding proteins (e.g., antibodies or fragments thereof) that compete for the same epitope means competition between antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., antibody or fragment thereof) tested (e.g., test antibody) prevents or inhibits (partially or fully) 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 2014PLoS One 9:e92451). Other assays that may 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); RIA is directly labeled using an I-125 labeled solid phase (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); RIA was directly labeled (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 100 x). Typically, when the competing antigen binding protein is present in excess, it will inhibit specific binding of the reference antigen binding protein to the co-antigen by at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%. In the case where each antigen binding protein (e.g., 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 said to "cross-compete" with each other to bind their respective epitope or "cross-block" each other. Typically, such cross-competition studies are conducted using the conditions and methods described above for competition studies, and the degree of blockage of each mode is at least 30%, at least 40%, or at least 50%. Additional methods and details of methods for identifying competitive 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). Affinity of molecule X for its partner Y is generally determined by the dissociation constant (K _d ) To represent. Affinity can be measured by conventional methods known in the art, including those described herein.

An "affinity matured" antibody refers to an antibody having one or more alterations in one or more hypervariable regions (HVRs) that result in an improvement in the affinity of the antibody for an antigen as compared to the parent antibody that does not have such alterations. In some examples, affinity matured antibodies refer to antibodies having one or more alterations in one or more Complementarity Determining Regions (CDRs) that result in an improvement in the affinity of the antibody for the antigen as compared to the parent antibody that does not have such alterations.

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 exhibits substantially background binding to non-target molecules. However, antigen binding proteins that specifically bind to a target antigen (e.g., ALPP and/or ALPPL 2) may cross-react with ALPP and/or ALPPL2 proteins from different species. In general, when the dissociation constant (K _D ) 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, the ALPP/ALPPL2 antigen binding protein specifically binds to human ALPP and/or ALPPL2, such as by Surface Plasmon Resonance (SPR) techniques (e.g., BIACore, GE-Healthcare Upps using antibodies as ligands and antigens as analytesala, sweden).

The term "K" as used herein _D "(M) refers to the dissociation equilibrium constant of a particular antigen binding protein-antigen interaction (e.g., antibody-antigen interaction). Affinity versus K, as used herein _D Inversely related such that higher affinity is intended to mean smaller K _D And lower affinity is intended to mean larger K _D 。

"antibody-drug-conjugate" or simply "ADC" refers to an antibody conjugated to a cytotoxic agent or cytostatic agent. The antibody-drug-conjugate typically binds to a target antigen (e.g., ALPP and/or ALPPL 2) on the surface of the cell, and then internalizes the antibody-drug-conjugate into the cell, releasing the drug in the cell.

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 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 self-consuming spacer:

the abbreviation "mc" refers to extended maleimidocaproyl:

the abbreviation "mp" refers to extended maleimide propionyl:

as used herein, a "PEG unit" is an organic moiety that includes repeating ethylene-oxy subunits (PEG or PEG subunits), and may be polydisperse, monodisperse, or discrete (i.e., having a discrete number of ethylene-oxy subunits). Polydisperse PEG is a heterogeneous mixture of size and molecular weight, whereas monodisperse PEG is generally purified from heterogeneous mixtures, thus providing a single chain length and molecular weight. Preferred PEG units include discrete PEG, compounds synthesized in a stepwise fashion rather than by a polymerization process. Discrete PEG provides a single molecule with a defined and specified chain length.

The PEG units provided herein comprise one or more polyethylene glycol chains, each polyethylene glycol chain comprising one or more ethyleneoxy subunits covalently linked to each other. Polyethylene glycol chains may be linked together, for example, in a linear, branched or star configuration. Typically, 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 urethane nitrogen of the methylene urethane unit (i.e., representing an example of R) prior to incorporation of the camptothecin conjugate. Typically, the terminal ethyleneoxy subunit in each polyethylene glycol chain that does not participate in covalent attachment to the remainder of the linker unit is capped with a PEG (typically an optionally substituted alkyl group such as-CH ₃ 、CH ₂ CH ₃ Or CH (CH) ₂ CH ₂ CO ₂ H) And (5) modification. Preferred PEG units have a single polyethylene glycol chain with 2 to 24 covalently linked-CH in tandem ₂ CH ₂ An O-subunit and is capped at one end with a PEG capping unit.

By "cytotoxic effect" is meant the depletion, elimination and/or killing of target cells.

By "cytotoxic agent" is meant an agent that has a cytotoxic effect on cells.

"cytostatic effect" means inhibition of cell proliferation.

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

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains 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 carboxy terminus of the heavy chain. However, the C-terminal lysine (Lys 447) of the Fc region may or may not be present. Unless otherwise indicated herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, md., 1991.

The "functional Fc region" has the "effector function" of the 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); down-regulation of cell surface receptors (e.g., B cell receptors; BCR), and the like. Such effector functions typically 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.

The "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 that differs from the native sequence Fc region by 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, the fcγr is a native human FcR. In some embodiments, the FcR is one that binds an IgG antibody (gamma receptor) and includes receptors of the fcγri, fcγrii and fcγriii subclasses (including allelic variants and alternatively spliced forms of these receptors). Fcyrii receptors include fcyriia ("activating receptor") and fcyriib ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor fcyriia contains an immune receptor tyrosine-based activating motif (ITAM) in its cytoplasmic domain. The inhibitory receptor fcyriib contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (see, e.g., daeron, annu. Rev. Immunol.15:203-234 (1997)). FcR is 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 for 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); down-regulation of cell surface receptors (e.g., B cell receptors); b cell activation. Such function may be affected, for example, by binding of the Fc effector domain to Fc receptors on immune cells having phagocytic or lytic activity or by binding of the Fc effector domain to components of the complement system. In general, the effects mediated by Fc binding cells or complement components result in inhibition and/or depletion of CD 33-targeted cells. The Fc region of an antibody can recruit cells expressing Fc receptors (FcR) and juxtapose them to antibody-coated target cells. Cells expressing surface fcrs of IgG including fcyriii (CD 16), fcyrii (CD 32) and fcyriii (CD 64) can act as effector cells that disrupt IgG-coated cells. Such effector cells include monocytes, macrophages, natural Killer (NK) cells, neutrophils and eosinophils. Binding of IgG to fcγr activates Antibody Dependent Cellular Cytotoxicity (ADCC) or Antibody Dependent Cellular Phagocytosis (ADCP). ADCC by CD16 ⁺ Effector cells are mediated by secreted membrane pore forming proteins and proteases, while phagocytosis is mediated by CD32 ⁺ And CD64 ⁺ Effector cell mediation (see, e.g., fundamental Immunology, 4 th edition, paul et al, lippincott-Raven, N.Y.,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).

A "human effector cell" is a leukocyte that expresses one or more FcRs and performs effector functions. In certain embodiments, the cell expresses at least fcyriii and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMC), natural Killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells may be isolated from natural sources, such as from blood.

"antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to the cytotoxic mechanism by which the Fc region of an antibody that binds to an antigen on the cell surface of a target cell interacts with Fc receptors (FcR) present on certain cytotoxic effector cells, such as NK cells, neutrophils and macrophages. This interaction enables these cytotoxic effector cells to subsequently kill target cells with cytotoxins. Primary cells mediating ADCC NK cells express fcyriii only, whereas monocytes express fcyri, fcyrii and fcyriii. 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 assess ADCC activity of a target molecule, an in vitro ADCC assay may 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 assays include PBMCs and NK cells. ADCC activity of the target molecule can also be assessed in vivo, for example in animal models such as those disclosed in Clynes et al Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998). Additional polypeptide variants having altered Fc region amino acid sequences (polypeptides having variant Fc regions) and increased or decreased ADCC activity are described, for example, in 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 (C1 q) to the Fc region of antibodies (of the appropriate subclass) that bind to their cognate antigen on the target cell. This binding activates a series of enzymatic reactions, eventually forming pores in the target cell membrane and subsequently leading to cell death. Activation of complement may also result in deposition of complement components on the surface of target cells that promote ADCC by binding to complement receptors (e.g., CR 3) on leukocytes. To assess complement activation, CDC assays may be performed, for example, as described in Gazzano-Santoro et al, J.Immunol. Methods 202:163 (1996). Variants of polypeptides having altered amino acid sequences of the Fc region (such as polypeptides having variant Fc regions) and increased or decreased C1q binding capacity are described, for example, in U.S. Pat. No. 6,194,551b1, 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 simply "ADCP", refers to the process by which antibody-coated cells are wholly or partially internalized by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the Fc region of an Ig.

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

The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to a polymer of nucleotides of any length. Such nucleotide polymers may contain natural and/or unnatural 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., nucleic acid, plasmid, phage, or virus) used to transfer a nucleic acid molecule into a host cell. Vectors typically include nucleic acid molecules engineered to contain one or more cloned polynucleotides encoding one or more polypeptides of interest 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. The carrier may comprise 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 the polypeptide of interest, and/or one or more selectable marker genes. The term includes vectors that are self-replicating nucleic acid molecules and 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 useful for expressing a polypeptide of interest in a host cell.

The term "host cell" or "host cell line" is used interchangeably herein and refers to a cell or population of cells that may or may not be a recipient of a vector or isolated polynucleotide. The host cell may be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells such as yeast; a plant cell; insect cells. Non-limiting exemplary mammalian cells include, but are not limited to NSO cells,Cells (Crucell) and 293 and CHO cells, and derivatives thereof, such as 293-6E and DG44 cells, respectively. These terms refer not only to the original cell but also to the progeny of such a cell. Due to, for example, mutation or environmental influence, certain modifications mayCan occur in offspring. These progeny 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 a coding sequence to which it is linked. The nature of such control sequences may depend on the host organism. In particular embodiments, control sequences for prokaryotes may include promoters, ribosome binding sites, and transcription termination sequences. Eukaryotic control sequences may include, for example, promoters containing one or more transcription factor recognition sites, transcription enhancer sequences, and transcription termination sequences. "control sequences" may include leader sequences and/or fusion partner sequences.

As used herein, "operably linked" means that the components to which the term applies are in a relationship that allows them to perform their inherent functions under the appropriate conditions. For example, a control sequence in a vector that is "operably linked" to a protein coding sequence is linked thereto such 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 such 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 when the exogenous DNA is introduced into the cell membrane, the cell has been "transfected". Numerous transfection techniques are well known in the art and are disclosed herein. See, e.g., 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 may be used to introduce one or more exogenous DNA portions into a suitable host cell.

The term "transformation" refers to a change in the genetic characteristics of a cell, and when the cell is modified to contain new DNA or RNA, the cell is transformed. For example, cells are transformed by transfection, transduction or other techniques to introduce new genetic material, so that they are genetically modified from their native state. Following transfection or transduction, the transforming DNA may be recombined with the DNA of the cell by physical integration into the chromosome of the cell, or may be maintained temporarily as an episomal element without being replicated, or may be replicated separately as a plasmid. A cell is considered to have been "stably transformed" when the transforming DNA replicates as the cell divides.

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

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

As used herein, "disease" or "disorder" refers to a condition in need of treatment.

As used herein, "cancer" and "tumor" refer to the interchangeable terms of any abnormal cell or tissue growth or proliferation in an animal. As used herein, the terms "cancer" and "tumor" encompass solid cancers and blood/lymph cancers, and also encompass malignant, premalignant, and benign growths, such as dysplasia. Solid tumors are abnormal growths or tumors of tissue that do not normally contain cysts or liquid areas. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. 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 or uterine cancer, salivary gland cancer, renal cancer (kidney cancer), renal cancer (renal cancer), liver cancer, prostate cancer, vulval cancer, thyroid cancer, liver cancer, brain cancer, endometrial cancer, testicular cancer, cholangiocarcinoma, gallbladder cancer, gastric cancer, melanoma, and various types of head and neck cancer.

"tumor burden" also referred to as "tumor burden" 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 a 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 imaging techniques, such as ultrasound, bone scanning, computed Tomography (CT) or Magnetic Resonance Imaging (MRI) scanning, when in vivo.

The terms "metastatic cancer" and "metastatic disease" mean a cancer that has spread from a site of origin to another part of the body, for example to regional lymph nodes or to a distant site.

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

As used herein, "treatment" is a method for achieving a beneficial or desired clinical result. As used herein, "treating" encompasses any administration or application of a therapeutic agent to a disease in a mammal (including a human). Beneficial or desired clinical results include, but are not limited to, any one or more of the following: alleviating one or more symptoms, alleviating the extent of a disease, preventing or delaying the spread of a disease (e.g., metastasis, such as to the lung or lymph nodes), preventing or delaying the recurrence of a disease, delaying or slowing the progression of a disease, ameliorating a disease state, inhibiting the progression of a disease or disease, inhibiting or slowing the progression of a disease or its progression, preventing its progression and remission (whether partial or total). "treating" also includes reducing the pathological consequences of a proliferative disease.

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

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

In the context of infectious diseases, the term "treatment" includes any or all of preventing the growth, proliferation or replication of pathogens causing infectious diseases and ameliorating one or more symptoms of infectious diseases.

The term "inhibition" refers to a reduction or cessation of any phenotypic feature, or to a reduction or cessation of the occurrence, extent, or likelihood of that feature. "reduce" or "inhibit" refers to reducing, decreasing, or preventing activity, function, and/or amount as compared to a reference. In certain embodiments, "reducing" or "inhibiting" refers to the ability to cause an overall reduction of 20% or greater. In another embodiment, "reducing" or "inhibiting" refers to the ability to cause an overall reduction of 50% or greater. In yet another embodiment, "reducing" or "inhibiting" 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. The reference may be obtained from healthy and/or non-diseased samples. In some examples, the reference may be obtained from an untreated sample. In some examples, the reference is obtained from a non-diseased or untreated sample of the subject individual. In some examples, the reference is obtained from one or more healthy individuals who are not subjects or patients.

As used herein, "delay of progression of a disease" means delay, impediment, slowing, delay, stabilization, inhibition, and/or delay of progression of a disease (such as cancer). This delay may have different lengths of time, depending on the disease history and/or the individual receiving the treatment. As will be apparent to those of skill in the art, a sufficient or significant delay may actually encompass prophylaxis, as the individual does not develop a disease. For example, the progression of advanced cancers, such as metastasis, may be delayed.

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

As used herein, "inhibiting" a function or activity refers to reducing the function or activity when compared to the same condition other than the condition or parameter 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 a tumor in the absence of the antibody.

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

For example, for treatment of a tumor, in some embodiments, a therapeutically effective amount of an anti-cancer 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 the anti-cancer agent inhibits cell growth or tumor growth by up to 100% in a treated subject (e.g., one or more treated subjects) relative to an untreated subject (e.g., one or more untreated subjects). In other embodiments of the present disclosure, tumor regression may be observed 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.

A 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 anti-cancer agent to a subject at risk of developing cancer (e.g., a subject with a pre-cancerous condition) or a subject suffering from a recurrence of cancer. In some embodiments, the prophylactically effective amount completely prevents the development or recurrence of cancer. "inhibiting" the progression or recurrence of cancer means reducing the likelihood of progression or recurrence of the cancer, or preventing the progression or recurrence of the cancer altogether.

As used herein, "sub-therapeutic dose" means a dose of therapeutic compound that is lower than the usual or typical dose of therapeutic compound when administered alone for the treatment of a hyperproliferative disease (e.g., 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 of skill 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 may also be performed, for example, once, multiple times, and/or over one or more extended periods of time.

As used herein, the term "monotherapy" means that the anti-ALPP/ALPPL 2 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 or other agents that are administered to a subject with cancer to treat symptoms associated with the cancer, but not to treat underlying cancer itself, including, for example, inflammation, pain, weight loss, and general malaise, may be administered during a monotherapy period.

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

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

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 cessation of the administration of one or more other agents. For example, administration of two or more therapeutic agents is administered at intervals of greater than 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 more.

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, auristatin (auristatin), and derivatives of podophyllotoxin); cytotoxic antibiotics; compounds that impair or interfere with DNA expression or replication (e.g., DNA minor groove binders); a growth factor receptor antagonist and a cytotoxic or cytostatic agent.

The phrase "pharmaceutically acceptable" means that the substance or composition is chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or the subject being treated therewith.

The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a formulation which exists in a form which allows the biological activity of the active ingredient to be effective, and which is free of additional components which have unacceptable toxicity to the subject to whom the formulation is to be administered. Such formulations may be sterile.

By "pharmaceutically acceptable carrier" is meant a nontoxic solid, semisolid or liquid filler, diluent, encapsulating material, formulation aid or carrier that is conventional in the art for use with therapeutic agents, which together constitute a "pharmaceutical composition" for administration to a subject. The 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. Pharmaceutically acceptable carriers are suitable for the formulation employed.

The phrase "pharmaceutically acceptable salt" as used herein refers to pharmaceutically acceptable organic or inorganic salts of the compounds of the present invention. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate (glucaronate), gluconate, formate, benzoate, glutamate, methanesulfonate "mesylate", ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 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 be referred to as comprising another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Multiple charged atoms may be part of a pharmaceutically acceptable salt with multiple counter ions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counter ions.

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

Overview II

The present invention provides antibody-drug conjugates, including anti-ALPP antibodies 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 are particularly effective in killing alpp+ expressing cells. The invention also provides antibody-drug conjugates, including anti-ALPPL 2 antibodies conjugated to vcMMAE or dLAE-MMAE, which are particularly effective in killing alppl2+ expressing cells. In a preferred embodiment, the invention provides antibodies that bind to both ALPP and ALPPL2 conjugated to vcMMAE or dLAE-MMAE (anti-ALPP/ALPPL 2 antibodies), which are particularly effective in killing cells expressing both alpp+ and alppl2+. ALPP and ALPPL2 have both been demonstrated to be expressed in a variety of cancers, including ovarian, lung, endometrial, testicular, and gastric cancers.

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

anti-ALPP/ALPPL 2 antigen binding proteins, including fragments

Various antigen binding proteins are provided herein and described in more detail below. The antigen binding proteins disclosed herein generally comprise 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 Complementarity Determining Regions (CDRs) are embedded, grafted, and/or linked. In some antigen binding proteins, HVRs or CDRs are embedded, grafted, or linked into a "framework" region that orients the HVRs or CDRs such that the appropriate antigen binding properties of the HVRs or CDRs are achieved. In some embodiments, the antigen binding protein comprises one or more VH and/or VL domains.

In some antigen binding proteins, HVR or CDR sequences are embedded, grafted, or linked to a protein scaffold or other biocompatible polymer. In some embodiments, the antigen binding protein is an antibody or is derived from an antibody. Thus, provided antigen binding proteins 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 portions or fragments of each of the foregoing. Examples of antigen binding proteins provided herein as fragments include, but are not limited to, fab ', F (ab') ₂ scFv, and domain antibodies.

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

A. Exemplary antigen binding proteins, including fragments

In one embodiment, the antigen binding proteins of the invention include 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 proteins disclosed herein comprise CDR-H1, CDR-H2, CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 56-58 or 60-62, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NOS: 63-65 or 68-70, respectively. In another embodiment, the antigen binding proteins disclosed herein comprise CDR-H1, CDR-H2, CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 56-58, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NOS: 63-65, respectively, wherein the CDRs are determined by Kabat. In another embodiment, the antigen binding proteins disclosed herein comprise 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 defined by IMGT.

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

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

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

In another embodiment, the ABP comprises (a) a VH domain comprising at least one, at least two or all three VH CDR sequences selected from the group consisting of (i) CDR-H1 comprising the amino acid sequence SEQ ID NO: 56; (ii) CDR-H2 comprising amino acid sequence SEQ ID NO 57; and (iii) a CDR-H3 comprising the amino acid sequence 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 sequences are selected from (i) CDR-L1 comprising the amino acid sequence SEQ ID NO 63; (ii) CDR-L2 comprising the amino acid sequence SEQ ID NO. 64; and (iii) a CDR-L3 comprising the amino acid sequence SEQ ID NO:65, wherein the CDR is determined by Kabat, and provided 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 selected from the group consisting of (i) CDR-H1 comprising the amino acid sequence SEQ ID NO: 60; (ii) CDR-H2 comprising the amino acid sequence SEQ ID NO 61; and (iii) a CDR-H3 comprising the amino acid sequence 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 sequences are selected from (i) CDR-L1 comprising the amino acid sequence SEQ ID NO. 68; (ii) CDR-L2 comprising the amino acid sequence SEQ ID NO 69; and (iii) CDR-L3 comprising the amino acid sequence SEQ ID NO 70, wherein the CDR is defined 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) CDR-H1 comprising the amino acid sequence SEQ ID NO:56 or SEQ ID NO: 60; (b) CDR-H2 comprising the amino acid sequence SEQ ID NO 57 or SEQ ID NO 61; and (c) CDR-H3 comprising the amino acid sequence SEQ ID NO 58 or SEQ ID NO 62, (d) CDR-L1 comprising the amino acid sequence SEQ ID NO 63 or SEQ ID NO 68; (e) CDR-L2 comprising the amino acid sequence SEQ ID NO 64 or SEQ ID NO 69; and (f) CDR-L3 comprising the amino acid sequence SEQ ID NO 65 or SEQ ID NO 70.

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

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

Some ABPs comprise a VH comprising CDR-H1, CDR-H2 and CDR-H3, wherein the CDRs of the VH have a total of up to 1, 2, 3, 4 or 5 amino acid changes relative to the corresponding CDR reference sequences, and wherein the CDR-H1 reference sequence has the amino acid sequence SEQ ID NO:56 or SEQ ID NO:60, the CDR-H2 reference sequence has the amino acid sequence SEQ ID NO:57 or SEQ ID NO:61, and the CDR-H3 reference sequence has the amino acid sequence 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 change is a conservative amino acid substitution.

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 up to 1, 2, 3, 4 or 5 amino acid changes relative to the corresponding CDR reference sequences, and wherein the CDR-L1 reference sequence has the amino acid sequence SEQ ID NO:63 or SEQ ID NO:68, the CDR-L2 reference sequence has the amino acid sequence 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 certain of the foregoing embodiments, the change is a conservative amino acid substitution.

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 SEQ ID NO:56 or SEQ ID NO:60, the CDR-H2 reference sequence has the amino acid sequence SEQ ID NO:57 or SEQ ID NO:61, and the CDR-H3 reference sequence has the amino acid sequence 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 SEQ ID NO:63 or SEQ ID NO:68, the CDR-L2 reference sequence has the amino acid sequence 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 certain of the foregoing embodiments, the change is a conservative amino acid substitution.

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 to an amino acid sequence selected from any one of SEQ ID NOs 9-16, provided that the ABP retains the ability to bind ALPP and/or ALPPL 2. 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 ALPP and/or ALPPL 2. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in any 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 some of these embodiments, such substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FR).

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 to an amino acid sequence selected from any of SEQ ID NOS.22-33, provided that the ABP retains the ability to bind ALPP and/or ALPPL 2. 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 ALPP and/or ALPPL 2. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in any 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 some of these embodiments, such substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FR).

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 of SEQ ID NOS.22-33, provided that ABP retains the ability to bind ALPP and/or ALPPL 2.

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 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 SEQ ID NO. 30, provided that ABP retains the ability to bind ALPP and/or ALPPL 2.

The antigen binding protein in any of the preceding embodiments may be any form of antibody. Thus, the antigen binding proteins described in any of the above embodiments may be, for example, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, or chimeric antibodies, as well as any of the ALPP binding fragments described above, such as single chain antibodies, fab fragments, F (ab') fragments, or fragments produced from a Fab expression library. Antibodies can be of any immunoglobulin isotype (e.g., igG, igE, igM, igD, igA and IgY), class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2) or subclass.

In certain embodiments, ABPs having CDR and/or variable domain sequences described herein are antigen-binding fragments (e.g., human antigen-binding fragments) and include, but are not limited to, 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 a portion of: hinge region, CH1, CH2, CH3 and CL domain. The disclosure also includes antigen binding fragments comprising any combination of one or more variable regions with hinge regions, CH1, CH2, CH3, and CL domains.

ABPs may be monospecific, bispecific, trispecific or have greater multispecific. The multispecific antibodies may be specific for different epitopes of ALPP and/or ALPPL2, or may be specific for both ALPP and/or ALPPL2 as well as heterologous proteins. See, for example, PCT publication WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; tutt et al, 1991,J.Immunol.147:60 69; U.S. patent No. 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 carboxy terminus of the light chain and/or heavy chain, such as the C-terminal lysine of the heavy chain, may be deleted or derivatized in some or all of the molecules in the composition. One specific example of such a modification is ABP with a carboxy-terminal lysine deletion of the heavy chain (e.g., as part of a post-translational modification). Furthermore, it should be understood that any of the sequences described herein include post-translational modifications to a particular sequence during ABP expression in a 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, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In another example, the chimeric antibody is a "class switch" antibody, wherein the class or subclass has been altered from that of the parent antibody. Some chimeric antibodies are described, for example, in U.S. Pat. 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 of the heavy and/or light chain variable regions described herein. In certain embodiments, the heavy and/or light chain variable domains are selected from, additional 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 proteins

In certain embodiments, ABP is a humanized antibody that binds ALPP and/or ALPPL 2. 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 a non-human "donor" antibody are grafted into human "acceptor" antibody sequences (see, e.g., queen, U.S. Pat. Nos. 5,530,101 and 5,585,089;Winter,US 5,225,539;Carter,US 6,407,213;Adair,US 5,859,205; and Foote, U.S. Pat. No. 6,881,557).

The acceptor antibody sequence may be, for example, a mature human antibody sequence, a complex of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. Human acceptor sequences with high sequence identity to the donor sequence in the variable region framework may be selected to match canonical forms and other criteria between the acceptor and donor HVRs or CDRs. Thus, a humanized antibody is an antibody having HVRs or CDRs entirely or substantially from a donor antibody and variable region framework sequences and constant regions (if present) entirely or substantially from a human antibody sequence. Similarly, a humanized heavy chain typically has all three HVRs or CDRs entirely or substantially from the donor antibody heavy chain, and heavy chain variable region framework sequences and heavy chain constant regions (if present) substantially from human heavy chain variable region framework and constant region sequences. Likewise, a humanized light chain typically has all three CDRs entirely or substantially from a donor antibody light chain, and light chain variable region framework sequences and light chain constant regions (if present) substantially from human light chain variable region framework and constant region sequences. An HVR or CDR in a humanized antibody is substantially from a corresponding HVR or CDR in a non-human antibody when at least 80%, 85%, 90%, 95%, or 100% of the corresponding residues between the corresponding HVRs or CDRs are identical (as defined by Kabat). When at least 80%, 85%, 90%, 95% or 100% of the corresponding residues 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 a human variable region framework sequence or a 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 prepared with fewer than all HVRs or CDRs (e.g., at least 3, 4, or 5) 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).

They may be selected for substitution based on the likely effect of certain amino acids from human variable region framework residues on HVR (e.g., CDR) conformation and/or binding to antigen. The study of this possible effect is empirically observed by modeling, examining the characteristics of amino acids at specific positions or the effect of substitution or mutagenesis of specific amino acids.

For example, when the amino acids between the murine framework residues of the murine variable region and the selected human variable region framework residues are different, the human framework amino acids may be substituted with equivalent framework amino acids from the mouse antibody, in which case the amino acids are reasonably expected:

(1) Non-covalent direct binding to the antigen(s),

(2) Adjacent to the HVR or CDR regions,

(3) Otherwise interact with the HVR or CDR regions (e.g., about in the regionInner);

(4) Mediate interactions between heavy and light chains, or

(5) Is the result of somatic mutation in the mouse chain.

(6) Is a glycosylation site.

(1) Framework residues of class- (3) are sometimes alternately referred to as canonical residues and vernier residues. Canonical residues are framework residues that define the canonical class of donor CDR loops that determine the conformation of the CDR loop (Chothia and Lesk, j.mol. Biol.196,901-917 (1987); thornton and Martin, j.mol. Biol.,263,800-815,1996). Vernier residues refer to the framework residues that support the antigen binding loop conformation and play a role in fine-tuning the antibody to 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 Franson, (2008) front. Biosci.13:1619-1633, and are further described, for example, in the following documents: 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) migration); padlan, (1991) mol. Immunol.28:489-498 (describing "re-surfacing"); dall' Acqua et al, (2005) Methods 36:43-60 (describe "FR shuffling"); and Osbourn et al, (2005) Methods36:61-68 and Klimka et al, (2000) Br.J.cancer,83:252-260 (describes the "guide selection" method of FR shuffling).

Human framework regions useful for humanization include, but are not limited to: the framework regions were selected using the "best fit" method (see, e.g., sims et al (1993) J.Immunol.151:2296); framework regions of consensus sequences of human antibodies derived from specific subsets of the 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, 151:2623); human mature (somatic mutation) 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 of the CDRs and/or heavy and/or light chain variable regions disclosed herein. Specific examples of such antibodies include humanized forms of the mouse antibody 12F 3. One such humanized variant of the mouse antibody 12F3 is designated 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. Humanized antibodies of the invention include variants of an HGLF humanized antibody wherein the humanized heavy chain mature variable region exhibits at least 90%, 95% or 99% identity to SEQ ID No. 15 and the humanized light chain mature variable region exhibits at least 90%, 95% or 99% sequence identity to 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 sequences shown in fig. 5 to 8.

D. Exemplary antibody constant regions

For those embodiments in which ABP is an antibody, the heavy and light chain variable regions of the antibodies described herein may be linked 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 kappa and lambda. In some embodiments, an antibody described herein comprises a human IgG constant region. In some embodiments, an antibody described herein comprises a human IgG4 heavy chain constant region. In some of these embodiments, the antibodies described herein comprise an S228P mutation in the human IgG4 constant region. In some embodiments, an antibody described herein comprises a human IgG4 constant region and a human kappa light chain.

In the present description and claims, unless explicitly stated or known to those skilled in the art, the numbering of residues in the heavy chain of immunoglobulins is that of the EU index as in Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition, 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 exhibit allotypic variation and allotypic variation of the same family between individuals, i.e., the constant regions may differ in one or more polymorphic positions among individuals. Homotypic isoforms differ from allotypes in that serum that recognizes a homotypic isoform binds to non-polymorphic regions of one or more other isoforms. References to human constant regions include constant regions having any natural allotype or any arrangement of residues occupying polymorphic positions in a natural allotype. Furthermore, up to 1, 2, 5 or 10 mutations, such as those noted above, may be present relative to the native human constant region to reduce fcγ receptor binding or increase binding to FcRn.

In some embodiments, one or more amino acids at the amino or carboxy terminus of the light chain and/or heavy chain, such as the C-terminal lysine of the 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 is desired. For example, human isotopes 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 induced stronger cell-mediated effector functions than human IgG2 and IgG 4. The light chain constant region may be lambda or kappa.

In addition, substitutions may be made in the constant region to reduce or increase effector functions, 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 extend half-life in humans (see, e.g., hinton et al, J. Biol. Chem.279:6213,2004), as described in more detail below.

E. Variants

The antigen binding proteins provided herein also include amino acid sequence variants of the antigen binding proteins provided herein. For example, variants with improved antibody binding affinity and/or other biological properties may be prepared. Amino acid sequence variants of antigen binding proteins can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antigen binding protein or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antigen binding protein. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, provided that the final construct has the desired properties, e.g., antigen binding.

1. Substitution, insertion and deletion variants

In some embodiments, the antigen binding proteins are variants having one or more amino acid substitutions, deletions, and/or insertions relative to the antigen binding proteins described herein. In certain such embodiments, the variants have 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, substitution of one amino acid in a polypeptide with another amino acid. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. Naturally occurring residues can be divided into several classes 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) Alkaline: his, lys, arg;

(5) Residues that affect chain orientation: gly, pro;

(6) Aromatic: trp, tyr, phe.

Target sites for substitution mutagenesis include CDRs and FR. Conservative substitutions are shown below under the heading "preferred substitutions" in table 2. More substantial changes are provided under the heading "exemplary substitutions" in table 2, and are described further below with respect to the amino acid side chain class. Amino acid substitutions may be introduced into the antibody of interest and the product screened for a desired activity, such as retention/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

TABLE 2

Original residue	Exemplary substitution	Preferably substituted
			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 (N-leucine)	Leu
			Leu	Norleucine; ile; val; met; ala; phe (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 (N-leucine)	Leu

Non-conservative substitutions involve replacing a member of one of these classes with a member of another class.

In altering the amino acid sequence of an antigen binding protein (e.g., an anti-ALPP/ALPPL 2 antibody), in some embodiments, the hydropathic index of amino acids may be considered. Each amino acid is assigned a hydrophilicity 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 hydrophilic amino acid index in conferring biological function on protein interactions is known in the art. Kyte et al, 1982, J.mol.biol.,157:105-131. It is known that certain amino acids may be substituted with other amino acids having similar hydrophilicity indices or scores and still retain similar biological activity. In making the change based on the hydropathic index, in certain embodiments, substitution of amino acids within ±2 of the hydropathic index is included. In certain embodiments, those amino acids that are within ±1 are included, and in certain embodiments, those amino acids that are within ±0.5 are included.

It will also be appreciated in the art that substitution of like amino acids may be effectively performed on the basis of hydrophilicity, particularly when the thus produced biologically functional protein or peptide (e.g., antibody) is intended for use in immunological embodiments, as in the case of the present invention. In certain embodiments, the maximum local average hydrophilicity of a protein (affected by the hydrophilicity of its neighboring amino acids) is related to its immunogenicity and antigenicity, i.e., to 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). In making the change based on similar hydrophilicity values, in certain embodiments, substitutions of amino acids having hydrophilicity values within ±2, in certain embodiments, those within ±1, and in certain embodiments, those within ±0.5 are included. Epitopes can also be identified from primary amino acid sequences based on hydrophilicity. These regions are also referred to as "epitope core regions".

Alterations (e.g., substitutions) may be made in the CDRs, for example, to increase antibody affinity. Such changes may be made in CDR "hot spots", i.e., residues encoded by codons that undergo high frequency mutations during somatic maturation (see, e.g., chordhury, methods mol. Biol.207:179-196 (2008)) and/or residues that contact antigen, and the resulting variant VH or VL tested for binding affinity. Affinity maturation by constructing and re-selecting secondary libraries is described, for example, in Hoogenboom et al Methods in Molecular Biology 178:1-37 (edited by O' Brien et al, human Press, totowa, N.J. (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or mutagenesis to oligonucleotides). Then, a secondary library is created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves a method for CDRs in which 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, provided that such changes do not significantly reduce the ability of the antibody to bind to an antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) may be made in the CDRs that do not significantly reduce binding affinity. Such alterations may be, for example, outside of the antigen-contacting 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.

One method that may be used to identify residues or regions in an antibody that may be targeted for mutagenesis is referred to as "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a group of residues or target residues (e.g., charged residues such as arg, asp, his, lys and glu) are identified and substituted 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 may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively, or in addition, the crystal structure of the antigen-antibody complex may identify the point of contact between the antibody and the antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired property.

Amino acid sequence insertions include amino-and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusion of the N-or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a polypeptide that extends 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 having substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutants in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

In certain embodiments, antibody variants having improved or reduced binding to FcR 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, e.g., 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 of the human IgG1 Fc region resulted in IgG variants with altered FcgammaR binding affinity (Shields et al, 2001, J.biol. Chem. 276:6591-604). Subsets of these variants involving substitution to Ala at Thr256/Ser298, ser298/Glu333, ser298/Lys334 or Ser298/Glu333/Lys334 demonstrated increased binding affinity and ADCC activity for FcgammaR when compared to parent IgG1 (Shields et al, 2001, J.biol. Chem.276:6591-604; okazaki et al, 2004, J.mol. Biol. 336:1239-49).

In some embodiments, alterations are made in the Fc region to alter (i.e., improve or reduce) Clq binding and/or Complement Dependent Cytotoxicity (CDC), for example, 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 (C1 q binding and CDC activity) of an antibody can be increased by substitution at Lys326 and Glu333 (Idusogene et al, 2001, J. Immunol. 166:2571-2575). Identical substitutions in the human IgG2 backbone can convert antibody isotypes that bind poorly to C1q and have a severe lack of complement activation activity to antibody isotypes that bind both to C1q and mediate CDC (Idusogie et al, 2001, J.Immunol.166:2571-75). Several other methods have also been applied to increase the complement binding activity of antibodies. For example, grafting an 18 amino acid carboxy-terminal tail of IgM to the carboxy terminus of IgG greatly enhances their CDC activity. This was observed even for IgG4 which generally had no detectable CDC activity (Smith et al, 1995, J. Immunol. 154:2226-36). Furthermore, substitution of Ser444 located near the carboxy terminus of the heavy chain of IgG1 with Cys induces tail-to-tail dimerization of IgG1 with CDC activity 200 times that of monomeric IgG1 (Shopes et al, 1992, J.Immunol.148:2918-22). In addition, bispecific diabody 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 heavy chain amino acid residues 318, 320 and 322 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, can also reduce or eliminate C1q binding by replacing any of the three residues. Ser, thr, cys and Met can be used for residues 320 and 322, rather than 318, to reduce or eliminate C1q binding activity. Substitution of the 318 (Glu) residue with a polar residue may alter, but does not eliminate, the C1q binding activity. Replacement of residue 297 (Asn) with Ala resulted in elimination of cleavage activity, but only a slight decrease in affinity for C1q (about one third of the original). This change disrupts the glycosylation site and the presence of carbohydrates required for complement activation. Any other substitution at this site will also disrupt the glycosylation site. The following mutations, and any combination thereof, also reduce C1q binding: D270A, K322A, P a and P311S (see WO 06/036291).

The half-life of an antibody provided herein can be increased or decreased to alter its therapeutic activity. FcRn is a receptor similar in structure to MHC class I antigens non-covalently bound to β2-microglobulin. FcRn regulates IgG catabolism and their transcytosis across tissues (Gheti e and Ward,2000, annu. Rev. Immunol.18:739-766; ghetie and Ward,2002, immunol. Res. 25:97-113). IgG-FcRn interactions occur at pH 6.0 (pH of intracellular vesicles), but not at pH 7.4 (pH of blood); this interaction enables recirculation of IgG back into the circulation (Ghetie and Ward,2000, ann. Rev. Immunol.18:739-766; ghetie and Ward,2002, immunol. Res. 25:97-113). Human IgG has been mapped ₁ The region involved in FcRn binding (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 a longer serum half-life. Thus, with unmodified IgG ₁ In contrast, these modified IgG ₁ Molecules may be able to perform their effector functions for a longer period of time 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 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 a substitution of Fc region residue 434 (see, e.g., U.S. patent 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 accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Natural antibodies produced by mammalian cells typically comprise branched, double-antennary oligosaccharides, which are typically linked to Asn297 of the CH2 domain of the Fc region by an N-bond. 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, as well as fucose attached to GlcNAc in the "stem" of a double-antennary oligosaccharide structure.

Engineering this glycoform on IgG can significantly improve IgG-mediated ADCC. The addition of bisected N-acetylglucosamine modifications to this glycoform (Umana et al, 1999, nat. Biotechnol.17:176-180; davies et al, 2001, biotech. Bioeng.74:288-94) or the removal of fucose from this 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 that improve the binding between IgG Fc and FcγR, thereby enhancing Ig-mediated ADCC activity. Antibodies comprising such substitutions or engineering are included in some embodiments provided herein.

In certain embodiments, antibodies are provided having a carbohydrate structure that lacks fucose (directly or indirectly) attached to an Fc region. For example, the amount of fucose in such antibodies 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. complex, hybrid and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, as described for example in WO 2008/077546. Asn297 refers to an asparagine residue at about position 297 in the Fc region (EU numbering of Fc region residues); however, asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e. between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. See, for example, U.S. patent publication No. US2003/0157108 (Presta, l.); US2004/0093621 (Kyowa Hakko Kogyo Co., ltd.). Examples of publications related to "defucosylation" or "fucose deficiency" antibody variants include: US2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/015614; US2002/0164328; US2004/0093621; US 2004/013321; US 2004/010704; US2004/0110282; US2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; ok azaki 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 Ser. No. 2003/0157108A1, presta, L, and WO 2004/056312A 1, adams et al, especially in example 11), and knockout cell lines such as alpha-1, 6-fucosyltransferase genes, 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 WO 2003/085107).

Other antibodies are also provided that contain bisected oligosaccharides, for example wherein a double-antennary oligosaccharide linked to the Fc region of the antibody is bisected by GlcNAc. Such antibodies may have reduced fucosylation and/or improved ADCC function. Examples of such antibodies are described, for example, in WO 2003/01878 (Jean-Maiset et al), U.S. Pat. No. 6,602,684 (Umana et al) and U.S. Pat. No. 2005/0123946 (Umana et al). Antibodies having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in 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 comprise substitution of a natural amino acid for a cysteine residue at amino acid positions 234, 235, 237, 239, 267, 298, 299, 326, 330 or 332 in the human IgG1 isotype, preferably an S239C mutation (substitution of the constant region is according to the EU index). The presence of additional cysteine residues allows for the formation of interchain disulfide bonds. This inter-chain disulfide bond formation can cause steric hindrance, thereby reducing the affinity of the Fc region-fcγr binding interactions. Cysteine residues introduced in or near the Fc region of the IgG constant region can also be used as sites for conjugation to therapeutic agents (e.g., coupling cytotoxic drugs, such as maleimide derivatives of drugs, using thiol-specific reagents). The presence of the therapeutic agent causes steric hindrance, thereby further reducing the affinity of the Fc region-fcγr binding interactions. Other substitutions at any of positions 234, 235, 236 and/or 237 reduce the affinity for fcγ receptors, in particular fcγri receptors (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 a drug moiety or linker-drug moiety, to create an immunoconjugate as described further herein. In certain embodiments, any one or more of the following residues may be substituted with a cysteine: v205 of light chain (Kabat numbering); a118 (EU numbering) of 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

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

F. Competitive antigen binding proteins

Antigen binding proteins provided herein include those that compete with one of the above-described 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 to overlapping epitopes. In one embodiment, such ABPs bind to an epitope having the amino acid sequence of SEQ ID NO:73 and/or SEQ ID NO: 74. ABPs including fragments competing with the exemplary ABPs are expected to exhibit similar functional properties (e.g., one or more of the activities described above).

In some embodiments, ABPs provided include those that compete with antibodies having: (a) All 6 CDRs listed for the same antibody as in SEQ ID NOS: 56-58 or 60-62 and 63-65 or 68-70; (b) VH and VL listed for the same antibodies as in SEQ ID NOs 15 and 30; or (c) the light and heavy chains specified for the same antibodies as in SEQ ID NOS: 40 and 50.

G. Antigen binding proteins that bind the same epitope

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

One method for determining the epitope or epitope region to which a specific antibody binds ("epitope region" is a region comprising or overlapping an epitope) involves assessing the binding of ABP to a peptide comprising ALPP and/or ALPPL2 fragments (e.g., non-denatured or denatured fragments). A series of overlapping peptides comprising ALPP and/or ALPPL2 (e.g., human ALPP and/or human ALPPL 2) sequences can be prepared and screened for binding, for example, in a direct ELISA, a competitive ELISA (where the ability of the peptide to prevent binding of antibodies to ALPP and/or ALPPL2 bound to wells of a microtiter plate) or on a chip. Such peptide screening methods may not 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 chains.

In other embodiments, regions containing residues that are contacted with or buried by an antibody can be identified by mutating specific residues in ALPP and/or ALPPL2 and determining whether ABP can bind to the mutated or variant ALPP and/or ALPPL2 protein. By performing a plurality of separate mutations, residues that play a direct role in binding or are sufficiently close to the antibody can be identified such that the mutation can affect the binding between the antigen binding protein and the antigen. From knowledge of these amino acids, domains or regions of the antigen containing residues that are in contact with ABP or covered by antibodies can be elucidated. Such domains may include binding epitopes of ABP. The general approach to such scanning techniques involves substitution of amino acids in the wild-type polypeptide with arginine and/or glutamic acid residues (typically alone). These two amino acids are commonly used in such scanning techniques because they are charged and bulky and therefore have the potential to disrupt the binding between ABP and ALPP and/or ALPPL2 in the region of the ALPP and/or ALPPL2 into which the mutation is introduced. Arginine present in wild-type antigen is replaced with glutamic acid. A number of such individual mutants were obtained and the collected binding results were analyzed to determine which residues affected 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 of identifying epitopes is MS-based protein footprints such as hydrogen/deuterium exchange mass spectrometry (HDX-MS) and rapid 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.

Epitopes bound by ABPs can also be determined by structural methods such as X-ray crystal structure determination, molecular modeling and Nuclear Magnetic Resonance (NMR) spectroscopy, including NMR determination of H-D exchange rates of labile amide hydrogens in the antigen when free and when bound in complex with ABPs (see, e.g., zinn-just et al (1992) Biochemistry 31,11335-11347; and Zinn-just et al (1993) Biochemistry 32, 6884-6891).

The X-ray crystallographic analysis may be accomplished using any method known in the art. Examples of crystallization methods are described, for example, by Giege et al (1994) Acta crystal grogr.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 (university of Yersinia, 1992, issued by Molecular Simulations, inc.; see, e.g., bluntell and Johnson (1985) meth. Enzymol.114 and 115, H.W. Wyckoff et al, academic Press; U.S. patent application publication No. 2004/0014194) and BUSTER (Brinogne (1993) Acta Cryst. D49:37-60; brinogne (1997) meth. Enzymol.276A:361-423, carter and Sweet editor; roveri et al (2000) Acta Cryst. D56:1313-1323).

In some embodiments, ABP binds to a contiguous epitope. In a preferred embodiment, ABP binds to an epitope having the 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) may be a single polypeptide, or may comprise two, three, four, five, six, seven, eight, nine, or ten (identical or different) polypeptides. In some embodiments in which the antibody or antigen-binding fragment thereof is a single polypeptide, the antibody or antigen-binding fragment may 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 may be the same or different from each other (and may specifically bind the same or different antigen or epitope).

The different portions of the antigen binding proteins described herein may be arranged in various configurations to obtain additional antigen binding proteins. For example, in some embodiments in which the antibody or antigen-binding fragment is a single polypeptide, the first antigen-binding domain and the second antigen-binding domain (if present) may each be independently selected from: VH domains, VHH domains, VNAR domains, and scFv. In some embodiments in which the antibody or antigen-binding fragment is a single polypeptide, the antibody or antigen-binding fragment may 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 conjugate. Other examples of antigen binding domains that may be used when the antibody or antigen binding fragment is a single polypeptide are known in the art.

V _H The H domain is capable ofSingle monomer variable antibody domains found in camelids. V (V) _NAR The domain is a single monomer variable antibody domain that can be found in cartilaginous fish. V (V) _H H domain and V _NAR Non-limiting aspects of domains are described, for example, in some documents: cromine 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, nanomedicine10:161-174,2015; kovaleva et al, expert. Opin. Biol. Ther.14:1527-1539,2014; krah et al, immunology. 38:21-28,2016; mujic-Delic et al, trends 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. Invest. 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 in which 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 The domain, or at least one antigen binding domain, is V _NAR A 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, an 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 in which an 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 may be assembled (e.g., non-covalently assembled) to form one or more antigen-binding domains, e.g., an antigen-binding fragment of an antibody (e.g., any antigen-binding fragment of an antibody described herein), a VHH-scAb, a VHH-Fab, a bisfab, a F (ab') ₂ Diabody, crossMab, DAF (two-in-one), DAF (four-in-one), dutamab, DT-IgG, knob-in-hole normal light chain, knob-in-hole assembly, charge pair, fab-arm exchange, SEEDbody, LUZ-Y, fcab, kappa lambda-body, orthogonal Fab, DVD-IgG, igG (H) -scFv, scFv- (H) IgG, igG (L) -scFv, scFv- (L) -IgG, 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-CH 3, triad, 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, dock-lock, 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, 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 10 th extracellular domain of human fibronectin III), neomycin CBM4-2, lipocalin-derived antimonidin, engineered ankyrin repeat domain (DARPin), protein-a domain (protein Z), kunitz domain, im9, TPR protein, zinc finger domain, pVIII, GC4, transferrin, B domain of SPA, sac7d, a-domain, SH3 domain of Fyn kinase, and C-type lectin-like domain (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 are incorporated herein by reference in their entirety.

Antigen binding protein expression and production

A. Nucleic acid molecules encoding antigen binding proteins

Nucleic acid molecules encoding the antigen binding proteins described herein, or portions thereof, are also provided. Such nucleic acids include, for example: 1) Those encoding an antigen binding protein (e.g., an antibody or fragment thereof) or a derivative or variant thereof; 2) Polynucleotides encoding heavy and/or light chains, VH and/or VL domains, or 1 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) located within a variable domain; 3) Sufficient for use as hybridization probes, PCR primers, or sequencing primers to identify, analyze, mutate, or amplify such polynucleotides encoding the polynucleotides; 4) Antisense nucleic acids useful for inhibiting expression of such encoding polynucleotides; and 5) the aforementioned complementary sequences. The nucleic acid may be of any length. They may 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 may 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 acid may be single-stranded or double-stranded.

The nucleic acid molecule may be present in whole cells, cell lysates or in partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially pure" when purified from other cellular components or other contaminants such as other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA linked to naturally isolated DNA) or proteins by standard techniques including alkali/SDS treatment, csCl strips, column chromatography, restriction enzymes, agarose gel electrophoresis, and other techniques well known in the art. See F.Ausubel et al (1987) Current Protocols in Molecular Biology, greene Publishing and Wiley Interscience, new York. The nucleic acids described herein may be, for example, DNA or RNA, and may or may not contain intronic sequences. In certain embodiments, the nucleic acid is a cDNA molecule.

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

Thus, nucleic acid molecules, such as anti-ALPP/ALPPL 2 antibodies, comprising polynucleotides encoding one or more strands of ABP are provided. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding a heavy or light chain of ABP (e.g., an anti-ALPP/ALPPL 2 antibody). In some embodiments, the nucleic acid molecule comprises both a polynucleotide sequence encoding the heavy chain of ABP (e.g., an anti-ALPP/ALPPL 2 antibody) and a polynucleotide sequence encoding the light chain thereof. 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 a VH of one of the antibodies provided herein. In another embodiment, the nucleic acid comprises a polynucleotide encoding a VL of one of the antibodies provided herein. In another embodiment, the nucleic acid encodes a VH and a 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 amino acid sequences (e.g., a heavy and/or light chain amino acid sequence, or VH and/or VL amino acid sequence, as 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.

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 are also provided. Thus, for example, in certain embodiments, the nucleic acid comprises a nucleotide sequence encoding a heavy 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 may 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 operations, the nucleic acid encoding a VL or VH is operably linked to another nucleic acid encoding another polypeptide, such as an antibody constant region or flexible linker.

An isolated nucleic acid encoding a VH region may be converted to a full length heavy chain gene by operably linking the nucleic acid encoding the VH to another nucleic acid molecule encoding a heavy chain constant region (hinge, CH1, CH2, and/or CH 3). The sequences of human heavy chain constant region genes are known in the art (see, e.g., kabat, e.a. et al (1991) Sequences of Proteins of Immunological Interest, fifth edition, U.S. Pat. No. 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 may be an IgG1, igG2, igG3, igG4, igA, igE, igM or IgD constant region, such as an IgG1 region. For Fab fragment heavy chain genes, the nucleic acid encoding a VH may be operably linked to another nucleic acid molecule encoding only the heavy chain CH1 constant region.

An isolated nucleic acid molecule encoding a VL region can be converted to a full length light chain gene (as well as a Fab light chain gene) by operably linking the nucleic acid molecule encoding VL to another nucleic acid molecule encoding a light chain constant region CL. The sequences of human light chain constant region genes are known in the art (see, e.g., kabat, e.a. et al (1991) Sequences of Proteins of Immunological Interest, fifth edition, U.S. Pat. No. 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 may be a kappa or lambda constant region.

To generate scFv genes, nucleic acid fragments encoding VH and VL are operably linked to a nucleic acid sequence encoding a flexible linker (e.g., encoding an amino acid sequence (Gly ₄ -Ser) ₃ ) Such that the VH and VL sequences can be expressed as contiguous single chain proteins, wherein the VL and VH regions are linked by a flexible linker (see, e.g., bird et al (1988) Science 242:423-426; huston et al (1988) Proc.Natl. Acad. Sci. USA85:5879-5883; 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, e.g., a fragment that may be used as a probe or primer or a fragment encoding 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 probe may comprise a labeling group, such as a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes can be used to identify cells expressing the polypeptide.

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

If desired, the expression vector may also include a secretion signal peptide sequence operably linked to the coding sequence of interest, such that the expressed polypeptide may be secreted by the recombinant host cell to more easily isolate the polypeptide of interest from the cell.

Expression and cloning vectors of the invention will typically contain a promoter recognized by the host organism and operably linked to a molecule encoding a polypeptide. A large number of promoters recognized by a variety of potential host cells are well known. The promoter is removed from the source DNA by restriction enzyme digestion and the desired promoter sequence is inserted into the vector, and a suitable promoter is operably linked to DNA encoding the heavy, light or other components of the antibodies and antigen-binding fragments of the invention. Suitable promoters for use in yeast hosts are also well known in the art. The yeast enhancer is advantageously used together with a yeast promoter. Suitable promoters for mammalian host cells are well known and include, but are not limited to, promoters obtained from the genomes of viruses such as polyomaviruses, fowlpox viruses, adenoviruses (such as adenovirus serotype 2, 8 or 9), bovine papilloma viruses, avian sarcoma viruses, cytomegaloviruses, retroviruses, hepatitis b viruses, and simian viruses 40 (SV 40). 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: SV40 early promoter (Benoist and Chambon,1981,Nature 290:304-310); CMV promoter (Thornsen et al, 1984, proc. Natl. Acad. U.S. A.81:659-663); promoters contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al, 1980, cell 22:787-797); herpes thymidine kinase promoter (Wagner et al, 1981, proc. Natl. Acad. Sci. U.S. A.78:1444-1445); promoter and regulatory sequences from metallothionein genes (Prister et al, 1982,Nature 296:39-42); and prokaryotic promoters such as the beta-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 may be inserted into the same expression vector. For example, a nucleic acid encoding a light chain or variable region of an anti-ALPP/ALPPL 2 antibody may be cloned into the same vector as a nucleic acid encoding a heavy chain or variable region of an anti-ALPP/ALPPL 2 antibody. In such embodiments, the two nucleic acids may be separated by an Internal Ribosome Entry Site (IRES) and under the control of a single promoter, such that the light and heavy chains are expressed from the same mRNA transcript. Alternatively, the two nucleic acids may be under the control of two separate promoters such that the light and heavy chains are expressed from two separate mRNA transcripts. In some embodiments, nucleic acid encoding the anti-ALPP/ALPPL 2 antibody light chain or variable region is cloned into one expression vector and nucleic acid encoding the anti-ALPP/ALPPL 2 antibody heavy chain or variable region is cloned into a second expression vector. In such embodiments, the host cell may be co-transfected with two expression vectors to produce the whole antibodies or antigen-binding fragments of the invention.

B. Host cells

After constructing the vector and inserting one or more nucleic acid molecules encoding components of ABPs described herein into appropriate sites of the vector, the complete vector may be inserted into an appropriate host cell for amplification and/or polypeptide expression.

Thus, in another aspect, there is also provided a host cell comprising a nucleic acid molecule or vector such as described herein. In various embodiments, ABP heavy and/or anti-light chains 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 choice of an appropriate host cell depends on a variety of factors such as the desired level of expression, the modification of the polypeptide (such as glycosylation or phosphorylation) required or necessary for activity, and the ease of folding into a biologically active molecule.

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

Exemplary prokaryotic host cells include eubacteria such as gram-negative or gram-positive organisms, e.g., enterobacteriaceae (Enterobacteriaceae), such as Escherichia (e.g., E.coli), enterobacter (Enterobacter), erwinia (Erwinia), klebsiella (Klebsiella), proteus (Proteus), salmonella (Salmonella) (e.g., salmonella typhimurium (Salmonella typhimurium)), serratia (Serratia) (e.g., serratia marcescens (Serratia marcescans)), and Shigella), and Bacillus (such as Bacillus subtilis (B.subsuitis) and Bacillus licheniformis (B.lichenius)), pseudomonas (Pseudomonas) and Streptomyces (Streptomyces).

Yeast may also be used as host cells including, but not limited to, saccharomyces cerevisiae (S.cerevisiae), schizosaccharomyces pombe (S.pombe), or Kluyveromyces lactis (K.lactis).

A variety of mammalian cell lines may 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 CCL 61), DXB-11, DG-44, and Chinese hamster ovary cells/-DHFR (CHO, urlaub et al, proc.Natl. Acad. Sci. USA77:4216,1980); SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (subcloning 293 or 293 cells for growth in suspension culture, graham et al, J.Gen virol.36:59,1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse support cells (TM 4, mather, biol. Reprod.23:243-251, 1980); monkey kidney cells (CV 1 ATCC CCL 70); african green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); rat hepatocytes (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. Thus, in another aspect, there is also provided a method for producing ABP as described herein. Typically, such methods comprise culturing a host cell comprising one or more expression vectors as described herein in a medium under conditions that allow expression of ABPs encoded by the one or more expression vectors; ABP is recovered from the culture medium.

In some embodiments, ABP is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in 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

ABPs provided herein can be conjugated to a cytotoxic or cytostatic moiety (including pharmaceutically compatible salts thereof) to form a conjugate, such as an Antibody Drug Conjugate (ADC). Particularly suitable moieties for conjugation to ABPs (e.g., antibodies) 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, ABP (e.g., an anti-ALPP/ALPPL 2 antibody) can be conjugated to a cytotoxic agent, such as a chemotherapeutic agent or toxin (e.g., a cytostatic or cytocidal agent, e.g., abrin, ricin a, pseudomonas exotoxin, or diphtheria toxin). Examples of useful classes of cytotoxic agents include, for example, DNA minor groove binders, DNA alkylating agents, and tubulin inhibitors. Exemplary cytotoxic agents include, for example, auristatin, camptothecin, calicheamicin, duocarmycin, etoposide, maytansinoids (e.g., DM1, DM2, DM3, DM 4), taxanes, benzodiazepines Classes (e.g. pyrrolo [1, 4)]Benzodiazepine->Class, indolobenzodiazepine +.>Class and oxazolidine benzodiazepine->Class) and vinca alkaloids.

In one embodiment, ABP (e.g., an anti-ALPP/ALPPL 2 antibody) is conjugated to a prodrug converting enzyme. The prodrug converting enzyme may be recombinantly fused to or chemically conjugated to an antibody using known methods. Exemplary prodrug converting enzymes are carboxypeptidase G2, beta-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, beta-lactamase, beta-glucosidase, nitroreductase and carboxypeptidase A.

Techniques for conjugating therapeutic agents to proteins, particularly to antibodies, are 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 may 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 cleavage agent). In some aspects, the therapeutic agent is linked to the antibody through a cleavable linker that is sensitive to cleavage in the intracellular environment of the ALPP-expressing cancer cell, but substantially insensitive to extracellular environment, such that when the conjugate is internalized by the ALPP-expressing cancer cell (e.g., in the endosome, or in the lysosomal environment or in the cell membrane pocket environment, e.g., by means of pH sensitivity or protease sensitivity), the conjugate is cleaved from the antibody. In some aspects, the therapeutic agent may also be linked to the antibody through a non-cleavable linker.

Typically, the ADC comprises a linker region between the therapeutic agent and the anti-ABP (e.g., anti-ALPP/ALPPL 2 antibody). The linker is typically cleavable under intracellular conditions such that 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 pocket). The linker may be a peptidyl linker cleaved, for example, by an intracellular peptidase or protease, including lysosomal or endosomal proteases. Cleavage agents may include cathepsins B and D and plasmin (see, e.g., dubowchik and Walker, pharm. Therapeutics 83:67-123,1999). Most typically a peptidyl linker which can be cleaved by enzymes present in the ALPP expressing cells. For example, a peptidyl linker (e.g., a linker comprising a Phe-Leu or Val-Cit peptide) that can be cleaved by thiol-dependent protease cathepsin-B that is highly expressed in cancerous tissue can be used.

The cleavable linker may be pH sensitive, i.e. sensitive to hydrolysis at certain pH values. Typically, the pH sensitive linker is hydrolyzable under acidic conditions. For example, acid labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis aconitamides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in the lysosome can be used. (see, e.g., U.S. Pat. Nos. 5,122,368;5,824,805;5,622,929; dubowchik and Walker, pharm. Therapeutic industries 83:67-123,1999; neville et al, biol. Chem.264:14653-14661,1989.) such linkers are relatively stable at neutral pH conditions (such as in blood), but are unstable below pH 5.5 or 5.0 (about the pH of the lysosome).

Other linkers are cleavable under reducing conditions (e.g., disulfide linkers). Disulfide linkages 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 edit, oxford U.S. Press,1987. See also U.S. Pat. No. 4,880,935.)

The linker may also be a malonate linker (Johnson et al, anti-icancer Res.15:1387-93, 1995), a maleimide benzoyl linker (Lau et al, biorg-Med-chem. 3:1299-1304, 1995) or a 3' -N-amide analogue (Lau et al, biorg-Med-chem. 3:1305-12, 1995).

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

Typically, the linker is substantially insensitive to extracellular environment, meaning that 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 when the ADC is present in the extracellular environment (e.g., in plasma). Whether the linker is substantially insensitive to the extracellular environment may be determined, for example, by: an equimolar amount of (a) ADC ("ADC sample") and (b) unconjugated antibody or therapeutic agent ("control sample") are incubated independently of plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours), and then the amount of unconjugated antibody or therapeutic agent present in the ADC sample is compared to the amount present in the control sample, as measured, for example, by high performance liquid chromatography.

The linker may also promote cellular internalization. When conjugated to a therapeutic agent (i.e., in the context of a linker-therapeutic moiety of an ADC or ADC derivative as described herein), the linker may promote cellular internalization. Alternatively, the linker may promote cellular internalization when conjugated to both the therapeutic agent and the antigen binding protein (e.g., an anti-ALPP/ALPPL 2 antibody) (i.e., in the context of an ADC as described herein).

Exemplary antibody-drug conjugates include auristatin-based antibody-drug conjugates, which means that the drug component is an auristatin drug. Auristatin binds to tubulin, has been shown to interfere with microtubule dynamics and cell nucleus and cell division, and has anticancer activity. Typically, the auristatin-based antibody-drug conjugate comprises a linker between the auristatin drug and ABP (e.g., anti-ALPP/ALPPL 2 antibody). The linker may be, for example, a cleavable linker (e.g., a peptidyl linker) or a non-cleavable linker (e.g., a linker released by antibody degradation). The auristatin may be auristatin E or a derivative thereof. The auristatin may be, for example, an ester formed between auristatin E and a keto acid. 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 is described in U.S. publication nos. 7,659,241, 7,498,298, 2009-011756, 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), as shown below, wherein Ab is ABP (e.g., anti-ALPP/ALPPL 2 antibody as described herein) and val-cit (vc) represents valine-citrulline dipeptide, and dLAE represents D-leucine-alanine-glutamic acid tripeptide:

Tripeptide:

/>

or a pharmaceutically acceptable salt thereof. Drug loading is represented by p, the number of drug-linker moieties per antibody. Depending on the context, p may represent the average number of drug-linker moieties per antibody in the antibody composition, also referred to as the average drug loading. P ranges from 1 to 20 and preferably from 1 to 12 or from 1 to 8. In some preferred embodiments, when p represents an average drug load, p ranges from 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, ABPs (e.g., anti-ALPP/ALPPL 2 antibodies) are linked to the drug-linker through cysteine residues of the antibody. In some embodiments, the cysteine residue is a cysteine residue engineered into an antibody. In other aspects, the cysteine residue is an interchain disulfide cysteine residue.

VI therapeutic application

A. Methods of treating diseases

In another aspect, methods of treating a disorder (e.g., cancer) associated with ALPP and/or ALPPL2 expressing cells 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. Thus, certain embodiments relate to treating a subject, such as a subject having cancer, using ABPs described herein (e.g., anti-ALPP/ALPPL 2 antibodies) as naked antibodies or as conjugates (e.g., antibody drug conjugates). In some of these embodiments, the method comprises administering to a subject in need thereof an effective amount of ABP (e.g., an anti-ALPP/ALPPL 2 antibody) or conjugate (e.g., an anti-ALPP/ALPPL 2 ADC) or a composition comprising such ABP or conjugate. In certain exemplary embodiments, the method comprises treating cancer in a cell, tissue, organ, animal or patient. Most typically, the method of treatment comprises treating cancer in a human. In some embodiments, the 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 therapeutic effect of Cancer can be measured in a number 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 RECIST 1.1 criteria. In some embodiments, the treatment achieved by the therapeutically effective amount is any one of inhibiting further tumor growth, inducing tumor regression, partial Response (PR), complete Response (CR), progression Free Survival (PFS), disease Free Survival (DFS), objective Response (OR), OR total survival (OS). In some embodiments, the treatment delays or prevents the occurrence of metastasis. Various methods can be used to monitor the progress of the treatment. For example, inhibition may result in a decrease in tumor size and/or a decrease in intratumoral metabolic activity. Both parameters can be measured by means of, for example, MRI or PET scanning. Inhibition can also be monitored by biopsy to determine levels of necrosis, tumor cell death, and intratumoral vascularity. The dosage regimen of the therapies described herein that is effective to treat a cancer patient can vary depending on factors such as the disease state, age and weight of the patient, and the ability of the therapy to elicit an anti-cancer response in the subject. While embodiments of the methods of treatment, medicaments and uses of the invention may not be effective in achieving a positive therapeutic effect in each 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 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 definition set forth in Eisenhauer et al, eur. Jcancer 45:228-247 (2009) for target lesions or non-target lesions, as the case may be, based on the context of the measured response.

An effective amount of ABP (e.g., an anti-ALPP/ALPPL 2 antibody) or ADC may be administered in one or more administrations, applications or doses and is not intended to be limited to a particular formulation or route of administration. Typically, a therapeutically effective amount of the active ingredient is in the range of 0.1mg/kg to 100mg/kg, for example 1mg/kg to 100mg/kg, 1mg/kg to 10mg/kg.

Exemplary doses of ABP (e.g., anti-ALPP/ALPPL 2 antibody) are, for example, 0.1mg/kg to 50mg/kg of patient body weight, more typically 1mg/kg to 30mg/kg, 1mg/kg to 20mg/kg, 1mg/kg to 15mg/kg, 1mg/kg to 12mg/kg, or 1mg/kg to 10mg/kg1, or 2mg/kg to 30mg/kg, 2mg/kg to 20mg/kg, 2mg/kg to 15mg/kg, 2mg/kg to 12mg/kg, or 2mg/kg to 10mg/kg, or 3mg/kg to 30mg/kg, 3mg/kg to 20mg/kg, 3mg/kg to 15mg/kg, 3mg/kg to 12mg/kg, or 3mg/kg to 10mg/kg.

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

The dosage administered may vary depending upon known factors such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; age, health, 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 may be increased beyond the upper level limit to quickly reach the desired blood or tissue level. Alternatively, the initial dose may be less than optimal and the daily dose may be gradually increased over the course of treatment.

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

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

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

Exemplary solid tumors that can be treated include, but are not limited to, malignant tumors of various 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, esophageal, gastric, hepatic, pancreatic, small intestine, colon and rectal, anal canal), genitalia and genitourinary tract (e.g., kidney, urothelium, bladder, ovary, uterus, cervix, endometrium, prostate, testis), skin (e.g., melanoma), and the like. 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 surgically removed (unresectable).

In certain embodiments, the 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 disorders, such as end-stage liver disease. 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, and the like. In some embodiments, the solid tumor associated with ascites is selected from the group consisting of breast cancer, colon cancer, pancreatic cancer, gastric cancer, uterine cancer, and ovarian cancer. In some embodiments, the cancer is associated with pleural effusion, such as lung cancer.

In particular embodiments, the cancer is HGSOC, wherein HGSOC progresses or recurs in the patient within six months after prior platinum-containing chemotherapy, and the patient has received one to three lines of prior anti-cancer therapy, including at least one line therapy comprising bevacizumab or a biological analog of bevacizumab. 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 modalities. In such a combination therapy method, two (or more) different treatments are delivered to a subject during the course of the subject afflicted with a disorder such that the effects of the treatments on the patient overlap at some point in time. In certain embodiments, delivery of one treatment still occurs at the beginning of delivery of a second treatment, such that there is overlap in administration. This is sometimes referred to herein as "simultaneous" or "parallel delivery. In other embodiments, the delivery of one therapy ends before the delivery of another therapy begins. In some embodiments of either case, the treatment is more effective due to the combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is observed with fewer second treatments, or the second treatment reduces symptoms to a greater extent, or a similar situation is observed with the first treatment, than if the second treatment was administered in the absence of the first treatment. In some embodiments, the delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than that observed when one treatment is delivered in the absence of another treatment. The effect of the two treatments may be partial addition, complete addition, or greater than addition (i.e., synergistic response). The delivery may be such that the effect of the delivered first therapy is still detectable when the second therapy is delivered.

In certain embodiments, the methods provided herein comprise administering to a subject an ABP (e.g., an anti-ALPP/ALPPL 2 antibody) or an ADC, e.g., a composition or formulation, as described herein, in combination with one or more additional therapies, e.g., surgery, radiation therapy, or administration of another therapeutic formulation. For example, in some embodiments, 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 immunomodulatory agent (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., anti-ALPP/ALPPL 2 antibody) or ADC and additional therapy may be administered simultaneously or sequentially.

In some embodiments, exemplary cytotoxic agents that may be used in combination with 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, MK 0683), entinostat (MS-275), panobinostat (LBH 589), trichostatin a (TSA), mo Xinuo stat (MGCD 0103), belinostat (PXD 101), romidepsin (FK 228, depsipeptide)), DNA methyltransferase inhibitors, nitrogen mustards, nitrosoureas, ethyleneimine, alkyl sulfonates, triazenes, folic acid analogs, nucleoside analogs, ribonucleotide reductase inhibitors, vinca alkaloids, taxanes, epothilones, intercalators, agents capable of interfering with signal transduction pathways, agents that promote apoptosis and radiation, or antibody molecule conjugates that bind surface proteins to deliver agents. In one embodiment, the cytotoxic agent that can be administered with the ABPs 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, emedine, mitomycin, etoposide, teniposide, vincristine, vinblastine, vinorelbine, colchicine, anthracyclines (e.g., doxorubicin or epirubicin), daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin D, doxorubicin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, or a maytansinoid.

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 ABP 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 present on tumor cells, such as Her2, CDC20, CDC33, mucin-like glycoproteins, and Epidermal Growth Factor Receptor (EGFR), and optionally induce cytostatic and/or cytotoxic effects on tumor cells displaying these proteins. Exemplary antibodies also include (trastuzumab) useful for the treatment of breast cancer and other forms of cancer, and +.>(rituximab),(Acidimo)Mab) and (I) of (L)>And->(epalizumab) which is useful for the treatment of non-hodgkin lymphomas and other forms of cancer. Other exemplary antibodies include panitumumab(IMC-C225)；ertinolib(Iressa)；/>(iodate 131 tositumomab); KDR (kinase domain receptor) inhibitors; anti-VEGF antibodies and antagonists (e.g., +.>Moti Sha Ni and vegff-TRAP); anti-VEGF receptor antibodies and antigen-binding regions; anti-Ang-1 and Ang-2 antibodies and antigen binding regions; antibodies to Tie-2 and other Ang-1 and Ang-2 receptors; tie-2 ligand; antibodies to Tie-2 kinase inhibitors; hif-1a and->(alemtuzumab) inhibitors. In certain embodiments, the cancer therapeutic agent is a polypeptide that selectively induces apoptosis in tumor cells, including but not limited to 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 (which include, but are not limited to, antibodies and antigen binding regions); soluble in water A sex TWEAK receptor antagonist (Wiley, U.S. patent No. 6,727,225); ADAM deaggregation domains to antagonize binding of integrins to their ligands (Fanslow et al, U.S. publication No. 2002/0042368); anti-eph receptors 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 VEGF, or soluble VEGF receptors or ligand binding regions thereof), such as +.>Or VEGF-TRAP ^TM The method comprises the steps of carrying out a first treatment on the surface of the And an anti-VEGF receptor agent (e.g., an antibody or antigen-binding region that specifically binds thereto); EGFR inhibitors (e.g., antibodies or antigen binding regions that specifically bind thereto), such as panitumumab,/or the like>(gefitinib), ->(erlotinib); anti-Ang-1 and anti-Ang-2 agents (e.g., antibodies or antigen-binding regions that specifically bind to or are a receptor for thereof, e.g., tie-2/TEK); and anti-Tie-2 kinase inhibitors (e.g., antibodies or antigen binding regions that specifically bind to and inhibit growth factor activity, 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 may be used in combination with the antigen binding protein 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, an "immune checkpoint molecule" refers to a molecule in the immune system that upregulates a signal (stimulatory molecule) and/or downregulates a signal (inhibitory molecule). Many cancers evade the immune system by inhibiting T cell signaling. In some embodiments of the present invention, in some embodiments, exemplary immune checkpoint molecules that may 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-activating gene 3 (LAG-3), carcinoembryonic antigen-associated cell adhesion molecule 1 (CEACAM-1), CEACAM-5, T cell activated V domain Ig inhibitor (VISTA), B and T lymphocyte attenuation factor (BTLA), T cell immunoreceptor with Ig and ITIM domains (TIGIT), leukocyte-associated immunoglobulin-like receptor 1 (LAIR 1), CD160, TGFR, adenosine 2A receptor (A2 AR), B7-H3 (also known as CD 276), B7-H4 (also known as VTCN 1), indoleamine 2, 3-dioxygenase (IDO), 2B4, killer cell immunoglobulin receptor (OX) 40, BBR 1, BBB 4-BB 1, ICT 4, ICL 3-ICH 3, and CD-co-stimulatory enzyme signaling factor (ICL) and CD-4/iL-mediated by the enzyme, and the enzyme type of the enzyme, ICP.

In certain embodiments, specific examples of immune checkpoint inhibitors that may be used in combination with ABP include, but are not limited to, the following monoclonal antibodies: PD-1 inhibitors such as palbociclizumabMerck) and nal Wu Liyou mab (/ -a)>Bristol-Myers Squibb); PD-L1 inhibitors such as Abilizumab (>Genentech), avermectin (a. Sup.)>Pfizer), dewaruzumab (>AstraZeneca); and CTLA-1 inhibitors such as ipilimumab (/ -)>Bristol-Myers Squibb) and tremelimumab (AstraZeneca).

Diagnostic applications

In another aspect, ABPs (e.g., anti-ALPP/ALPPL 2 antibodies or fragments thereof), polypeptides, and nucleic acids as provided herein can be used in methods of detecting, diagnosing, and monitoring diseases, disorders, or conditions associated with ALPP and/or ALPPL 2.

In some embodiments, the method comprises detecting expression of ALPP and/or ALPPL2 in a sample obtained from a subject suspected of having a disorder associated with ALPP and/or ALPPL 2. 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 the reference or comparative sample. In some embodiments, such methods can be used to determine whether an antibody or polypeptide described herein is a suitable treatment for a subject.

For example, in one embodiment, a cell or cell/tissue lysate is contacted with an anti-ALPP/ALPPL 2 antibody and binding between the antibody and the cell or antigen is determined. When a test cell exhibits 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 ALPPL 2. In some embodiments, the test cells are from human tissue.

Various methods known in the art for detecting specific antibody-antigen binding may be used. Exemplary immunoassays that can be performed according to the present invention include Fluorescence Polarization Immunoassays (FPIA), fluorescence Immunoassays (FIA), enzyme Immunoassays (EIA), turbidity suppressing immunoassays (NIA), enzyme linked immunosorbent assays (ELISA), and Radioimmunoassays (RIA).

Diagnostic applications provided herein include detection using ABP (e.g., anti-ALPP/ALPPL 2 antibodies or fragments thereof)Expression of ALPP and/or ALPPL2 and binding of ligands to ALPP and/or ALPPL 2. For diagnostic applications, ABPs are typically labeled with a detectable label group. Suitable labelling groups include, but are not limited to, the following: the radioisotope or radionuclide (e.g., ³ H、 ¹⁴ C、 ¹⁵ N、 ³⁵ S、 ⁹⁰ Y、 ⁹⁹ Tc、 ¹¹¹ In、 ¹²⁵ I、 ¹³¹ i) A fluorescent group (e.g., FITC, rhodamine, lanthanide phosphor), an enzymatic group (e.g., horseradish peroxidase, β -galactosidase, luciferase, alkaline phosphatase), a chemiluminescent group, a biotin group, or a predetermined polypeptide epitope recognized by a second reporter gene (e.g., leucine zipper pair sequence, binding site of a secondary antibody, metal binding domain, epitope tag). In some embodiments, the labeling group is coupled to ABP via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and may be used. Examples of methods that can be used to detect the presence of ALPP and/or ALPPL2 include immunoassays as described above.

ABP, on the other hand, can be used to identify one or more cells expressing ALPP and/or ALPPL 2. In a specific embodiment, the antigen binding protein is labeled with a labeling group and 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 (e.g., anti-ALPP/ALPPL 2 antibodies or fragments thereof) can also be used as staining reagents in pathology using techniques well known in the art.

Pharmaceutical composition and formulation

Also provided are pharmaceutical compositions comprising ABPs (e.g., anti-ALPP/ALPPL 2 antibodies or fragments thereof) and can be used in any of the therapeutic applications 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 dosages and concentrations employed. The pharmaceutical composition may be formulated as a liquid, frozen or lyophilized composition.

In certain embodiments, the pharmaceutical composition may contain formulation materials for altering, maintaining, or preserving the composition, such as pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeation. Suitable formulation materials include, but are not limited to, amino acids; an antimicrobial agent; an antioxidant; a buffering agent; a compatibilizer; a chelating agent; complexing agent; a filler; carbohydrates, such as mono-or disaccharides; a protein; coloring agents, flavoring agents, and diluents; an emulsifying agent; a hydrophilic polymer; a low molecular weight polypeptide; salt-forming counterions (such as sodium); a preservative; solvents (such as glycerol, propylene glycol or polyethylene glycol); sugar alcohols; a suspending agent; a surfactant or wetting agent; a stability enhancer; a tension enhancer; a delivery vehicle; and/or pharmaceutical adjuvants. Additional details and choices of suitable agents that may be incorporated into the pharmaceutical compositions are provided, for example, in Remington's Pharmaceutical Sciences, 22 nd edition, (Loyd v. Allen edit) Pharmaceutical Press (2013), ansel et al, pharmaceutical Dosage Forms and Drug Delivery Systems, 7 th edition, lippencott Williams and Wilkins (2004) and Kibbe et al, handbook of Pharmaceutical Excipients, 3 rd edition, pharmaceutical Press (2000).

The choice of the components of the pharmaceutical composition depends on, for example, the intended route of administration, the form of delivery and the dosage required. See, e.g., remington's Pharmaceutical Sciences, 22 nd edition, (Loyd v. Allen edit) Pharmaceutical Press (2013). The composition is selected to affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the disclosed antigen binding proteins. The primary vehicle or carrier in the pharmaceutical composition may be aqueous or non-aqueous. For example, a suitable vehicle or carrier may be water for injection or a physiological saline solution. In certain embodiments, the antigen binding protein composition may be prepared for storage by mixing a selected composition of the desired purity with an optional formulation in the form of a lyophilized cake or aqueous solution. Furthermore, in certain embodiments, the antigen binding proteins may be formulated as lyophilizates using suitable excipients.

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. A preferred route of administration for antigen binding proteins (e.g., antibodies) is IV infusion. In another preferred embodiment, the formulation is administered by intramuscular or subcutaneous injection.

IX. kit/article

Kits comprising ABPs described herein are also provided. In one embodiment, such kits comprise one or more containers comprising antigen binding proteins (e.g., anti-ALPP/ALPPL 2 antibodies) or unit dosage forms and/or articles of manufacture. 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 unit doses are provided in disposable pre-filled syringes for injection. In various embodiments, the composition contained in the unit dose may comprise: brine; buffers, other formulation components, and/or in 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 upon addition of an appropriate liquid (e.g., sterile water).

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

Certain kits are, for example, used for diagnosing cancer and comprise a container containing an antigen binding protein (e.g., an anti-ALPP/ALPPL 2 antibody) and one or more reagents for detecting binding of the antigen binding protein to ALPP and/or ALPPL 2. Such reagents may include, for example, fluorescent labels, enzymatic labels, or other detectable labels. The reagent may also include secondary or tertiary antibodies or reagents, such as an enzymatic reaction for producing a product that can be visualized. In one embodiment, the diagnostic kit comprises one or more antigen binding proteins, in labeled or unlabeled form, in a suitable container, incubation reagents for an indirect assay, and a substrate or derivatizing agent for detection in such an assay, depending on the nature of the label.

Kits such as provided herein may be used for in situ detection. Some methods of using such kits include removing a histological specimen from a patient, and then combining a labeled antigen binding protein (e.g., an anti-ALPP/ALPPL 2 antibody) with a biological sample. Using such methods, not only the presence of ALPP or ALPP fragments and/or ALPPL2 fragments can be determined, but also the distribution of such peptides in the examined tissue (e.g., in the case of assessing cancer cell spread).

In another aspect, anti-idiotype antibodies (Id) that bind to antigen binding proteins (e.g., anti-ALPP/ALPPL 2 antibodies) are provided. Id antibodies can be prepared by immunizing animals of the same species and genotype as the source of the anti-ALPP/ALPPL 2 mAb with mAb from which the anti-Id was prepared. Immunized animals can typically recognize and respond to the idiotype determinants of an immunized antibody by producing antibodies (anti-Id antibodies) against these idiotype determinants.

The following examples, including 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 level

The cell lines described in the examples below are maintained in culture according to the American Type Culture Collection (ATCC) or the German Collection of microorganisms and cell cultures (DMSZ), the Japanese cancer research resource library (JCRB) or other known conditions.

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

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

Tumor tissue arrays are obtained from commercial sources. Frozen or Formalin Fixed and Paraffin Embedded (FFPE) tissues were purchased from USBiomax Inc. All samples were at Bond-Max ^TM The treatment was carried out on an automatic dyeing machine (Leica).

Frozen samples sectioned on slides were fixed with acetone for 10 minutes prior to staining. Slides were incubated with anti-ALPP/ALPPL 2 primary antibody (H17E 2; thermo; catalog number MA 1-20245). Isotype matched mouse IgG1 was used as a negative control for background staining. For automatic IHC staining we used the finer DAB kit (Leica, catalog number DS 9800). Slides were incubated with 5 μg/ml of mouse monoclonal primary antibody against ALPP for 45 min and with peroxabolist (Biocare Medical catalog number PXA 969M) reagent for 15 min, followed by 20 min pre-incubation with protein blocks (DAKO catalog number X0909). Using Bond ^TM Dewax solution (Leica, catalog number AR 9222) will be sectioned on slides at 72 ℃Dewaxed and rehydrated FFPE slides of (c). Bond using EDTA based ^TM Epitope repair solution 2 (Leica, cat. No. AR 9640) was subjected to antigen repair at 95 ℃ -100 ℃ for 20 min, and then incubated with anti-ALPP/ALPPL 2 primary antibody (25C 3 monoclonal Ab; an in-house developed mouse monoclonal) for 45 min at 1 μg/ml. Isotype matched mouse IgG2a was used as a negative control for background staining. For automatic IHC staining we used the finer DAB kit (Leica, catalog number DS 9800). Slides were incubated for 20 min with 1 μg/ml of mouse monoclonal antibody to ALPP mAb, followed by 45 min with protein blocks (DAKO accession number X0909). After development, the sections were counterstained with hematoxylin and covered with a coverslip. Slides were assessed and scored by pathologists and images were taken using an aprio slide scanner (Leica). The staining intensity was 0 to +3 minutes, and the frequency was expressed in quartiles (0-25, 26-50, 51-75, and 76-100). ALPP/ALPPL2 expression was found to be high in a variety of solid tumor indications including ovary, testis and endometrium, as shown in table 4. ALPP/ALPPL2 expression was also present in 25% of lung adenocarcinoma, gastric carcinoma and bladder carcinoma samples.

TABLE 4 Table 4

9

Example 2: lead antibody selection

Conjugation and in vitro cytotoxicity

Mice were immunized with recombinant full length ALPPL 2. Lymphocytes collected from the spleen and lymph nodes of ALPP antibody-producing mice 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 IgG-producing hybridoma clones were selected. Antibodies from this hybridoma series were screened on HEK293 cell lines expressing ALPP, ALPPL2, ALPI and ALPL using an iQue according to the manufacturer's instructions. Antibodies that were cross-reactive to ALPP and ALPPL2 (but not ALPI and ALPL) were evaluated as ADCs.

Various mouse anti-ALPP/ALPPL 2 monoclonal mouse antibodies were conjugated to 10-12 loaded MDpr-PEG (12) -gluc-MMAE or auristatin T, which exhibited bystander activity or no bystander activity, respectively. Conjugation methods are described in U.S. publication No. 2018/0092984.

The CAOV3 (ALPP), COV644 (ALPP+) and NCI-H1651 (ALPPL2++ ALPP+) tumor cells were incubated with ALPP/ALPPL2 Antibody Drug Conjugates (ADC) for 96 hours at 37 ℃. Human IgG ADC was used as a negative control. Cell viability was measured using a Cell Titer Glo according to the manufacturer's instructions. Fluorescence signals were measured on a Fusion HT fluorescence microplate reader (Perk in Elmer, waltham, mass.). Data were normalized to untreated cells and x50 values were calculated using Graph Pad software. As shown in fig. 1-2, the Ab subgroup exhibited lower x50 values, with both payloads indicating higher drug delivery capacity.

Flow cytometry and saturation binding assays

Specificity and binding affinity were assessed with HEK293 cells expressing cynomolgus ALPP, human ALPP, ALPPL2, ALPI and ALPL. Briefly, hundred thousand HEK293 cells expressing targets were transferred to 96-well plates. Cells were pelleted by centrifugation and resuspended in 100. Mu.L PBS+2% w/v BSA. After blocking, cells were resuspended in PBS+2% w/v BSA, with unlabeled monoclonal anti-ALPP/ALPPL 2 antibodies ranging in concentration from 8pM to 666nM, and incubated on ice for 30 min. Cells were washed twice in PBS and resuspended in R-PE labeled goat anti-human or anti-murine secondary antibodies (Jackson Immunoresearch, west Grove, PA) for 30 minutes on ice. The specificity of monoclonal antibodies for human and cynomolgus ALPP and ALPPL2 was confirmed by flow cytometry, but not for other members of the alkaline phosphatase family. Fluorescence was analyzed using an Attune NxT flow cytometer and the percentage of saturated fluorescence signal was used to determine the percentage of binding and then to calculate apparent K _D . Antibodies 1C7 and 12F3 showed the lowest K among top candidates _D As shown in fig. 3. However, while SG82-12F3 and SG84-1F7 showed similar affinities for their targets, SG82-12F3 showed higher saturation levels for ALPP than SG84-1F7, as shown in FIG. 4. Finally, the 12F3 antibody was selected for humanization based on its excellent ADC cytotoxicity and high affinity for ALPP/ALPPL2 with epitopes conserved with cynomolgus orthologs.

Example 3: humanization and binding studies

The humanized antibody was derived from the murine 12F3 antibody. Eight humanized heavy chains (HA-HH) and twelve humanized light chains (L1-LI) were prepared incorporating back mutations at different positions. In some cases, the back mutations match the murine germline, and in other cases they are not (as in the case of somatic mutations). Humanized heavy and light chains are paired. See sequence alignment of figures 5-8 and specific mutations of tables 5-8.

Table 5: humanized mutations in h12F3 variable weight (vH) chain variants

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

vH variants

30

37

48

49

73

78

93

% person

hvHA

94.0

hvHB

T

N

92.0

hvHC

T

L

A

N

90.0

hvHD

T

N

L

A

90.0

hvHE

T

L

A

N

L

A

88.0

hvHF

T

V

L

A

N

L

A

88.0

hvHG

T

V

L

A

N

L

A

88.0

hvHH

T

V

L

A

N

L

A

87.0

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

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

/>

An antibody designated HAL1 (an antibody having a heavy chain variable region designated vHA and a light chain variable region designated vL 1), an antibody designated HAL2 (an antibody having a heavy chain variable region designated vHA and a light chain variable region designated vL 2), an antibody designated HAL3 (an antibody having a heavy chain variable region designated vHA and a light chain variable region designated vL 3), an antibody designated HALA (an antibody having a heavy chain variable region designated vHA and a light chain variable region designated vLA), an antibody designated HALB (an antibody having a heavy chain variable region designated vHA and a light chain variable region designated vLB), an antibody designated HALC (an antibody having a heavy chain variable region designated vHA and a light chain variable region designated vLC), a light chain antibody antibodies named HALD (antibodies with heavy chain variable region named vHA and light chain variable region named vLD), antibodies named HALE (antibodies with heavy chain variable region named vHA and light chain variable region named vLE), antibodies named HALE (antibodies with heavy chain variable region named vHA and light chain variable region named vLE), antibodies named HALF (antibodies with heavy chain variable region named vHA and light chain variable region named vLF), antibodies named mig (antibodies with heavy chain variable region named vHA and light chain variable region named vLG), antibodies named HALH (antibodies with heavy chain variable region named vHA and light chain variable region named vLH) and antibodies named HALI (antibodies with heavy chain variable region named vHA and light chain variable region named vLI) may be used in the present invention in place of HGLF antibodies. Similarly, antibodies having any arrangement of heavy chain variable regions designated vHA, vHB, vHC, vHD, vHE, vHF, vHG or vHH and light chain variable regions designated vL1, vL2, vL3, vLA, vLB, vLC, vLD, vLE, vLF, vLG, vLH or vLI may be used in place of HGLF antibodies in the present invention. 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 sequences are shown in FIGS. 5-8.

Humanized antibodies of low quality, low expression yield or unfavorable sequences were not evaluated in functional assays. Assessment of humanized antibody versus ALPPL2 expressing cells using flow cytometryApparent affinity. Briefly, K for each resulting antibody was then determined by a saturated binding assay _D . HEK293 cells stably expressing human ALPPL2 were aliquoted into 96-well v-plates at 1E5 cells per well. Each humanized ALPP/ALPPL2 antibody was added at concentrations of 0.2, 2 and 20nM and incubated on ice for 60 minutes. Cells were pelleted and washed 2 times with PBS/FBS, then 10 μg/ml of APC-labeled anti-human IgG mouse secondary antibody was added and incubated on ice for an additional 60 minutes. Cells were pelleted and washed 2 times with PBS/FBS and resuspended in 100 μl of 2% paraformaldehyde. Fluorescence was analyzed by flow cytometry, the percentage of saturation fluorescence signal was used to determine the percentage of binding and then apparent K was calculated based on the three antibody concentrations _D . Apparent K of recombinant humanized ALPP/ALPPL2 _D Comparison with c12F3 (chimeric 12F3IgG1 k) is shown in Table 9.

Table 9: determining binding (KD (nM)) of the hALPP-1 antibody variant on HEK-ALPPL2 cells by flow cytometry; nt=no test.

Example 4: h12F3 conjugation and in vitro cytotoxicity

Several h12F3 antibodies were constructed using hIGHV3-49/hIGHJ4 heavy chain variable region human germline and hIGKV1-33/hIGKJ 2 or hIGKV1D-43/hIGKJ2 or hIGKV1-16/hIGKJ2 light chain variable region human germline as human receptor sequences. These antibodies differ in the selection of amino acid residues that are mutated back to the mouse antibody or mouse germline sequence.

As mentioned above, humanized antibodies of low quality, low expression or unfavorable sequences 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 selection of potential antibody precursors, additional cytotoxicity evaluations were performed on different payloads, including conjugation of antibodies conjugated with 4-loaded mc-vc-PABC-MMAE or mp-dLAE-PABC-MMAE or with 8-loaded MDpr-PEG (12) -gluc-MMAE. Conjugation methods are 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/ALPPL 2 antibodies described herein as described in PCT/US2020/051648 (submitted 9, 18 days 2020). For conjugation with an antibody to mp-dLAE-PABC-MMAE, the antibody was partially reduced using the appropriate equivalent of TCEP (tris (2-carboxyethyl) phosphine) according to the procedure of US 2005/023849, which procedure is expressly incorporated herein by reference. Briefly, antibodies in phosphate buffered saline containing 2mM DTPA (pH 7.4) were treated with 2.1 equivalents of TCEP and then incubated at 37℃for about 45 minutes. thiol/Ab values were checked by reacting the reduced antibodies with compound 1 and determining the loading using hydrophobic interaction chromatography.

The tripeptide-based auristatin drug-linker mp-dLAE-PABC-MMAE compound was conjugated to a partially reduced antibody using the method of US 2005/023849, which method is expressly incorporated herein by reference. Briefly, drug-linker compounds (mp-dLAE-PABC-MMME) in DMSO (50% excess) were added to the reduced antibodies in EDTA-containing PBS and additional DMSO to obtain 10% -20% of total reaction co-solvent. After 30 minutes at ambient temperature, excess QuadraSil mp tm 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 ℃ until further use. The protein concentration of the resulting ADC composition was determined at 280 nm. The drug-to-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 prior to ADC treatment. Cells were treated with indicated doses of ADC and incubated for 96 hours at 37 ℃. In some experiments, non-antigen binding ADCs were included as negative controls. Cell viability of the 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 for 30 minutes at room temperature and luminescence was measured on an Envision microplate reader (Perkin Elmer, waltham, MA). As shown in fig. 9, humanized versions of h12F3 antibodies containing light chain F variants identified variants with high drug delivery capacity, especially when compared to other combinations.

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

Tumor cells were incubated with each ADC at 37℃for 96-144 hours. Non-binding (called h00 or IgG) ADCs were used as negative controls. Cell viability was measured using a Cell Titer Glo according to the manufacturer's instructions. Fluorescence signals were measured on a Fusion HT fluorescence microplate reader (Perkin Elmer, waltham, mass.). 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%). h12F3ADC implements single and two digits ng/ml IC in a set of cell lines with ALPP expression ranging from 30,000 to 500,000 ₅₀ Values.

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

By plotting the IC50 values of multiple cell lines, the cytotoxic efficacy of antibody drug conjugates using humanized variants of the same payload (mp-dLAE-MMAE) was compared. Humanized variants of the 12F3 antibody showed similar efficacy in vitro as shown in figure 10.

Based on (i) binding properties, (ii) ability to deliver drugs, and (iii) number of back mutations compared to other variants of the antibodies designated 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), it was finally selected as a lead humanized anti-ALPP/ALPPL 2 antibody (see tables 5 to 8).

Evaluation of tumor cancer cell spheroids by HGLF ADC was performed as follows: 100uL of cells were plated in ultra low adhesion round bottom 96 well plates (Corning, corning, NY) at 2.5E4 cells/well for 48 hours at 37 ℃. After incubation, 100uL of medium containing 2X ADC was added and incubated for 120 hours at 37 ℃. In some experiments, non-antigen binding ADCs were included as negative controls. Cell viability of the Cell lines was measured using a 3D Cell Titer Glo (Promega Corporation, madison, WI) according to the manufacturer's instructions. Cells were incubated with 3D Cell Titer Glo reagent for 30 minutes at room temperature and luminescence was measured on an Envision microplate reader (Perkin Elmer, waltham, MA). Results are reported as IC50, the concentration of compound required to produce a half maximal reduction in viability compared to vehicle treated cells (control = 100%). As shown in fig. 11 and table 11, h12F3 HGLF ADC conjugated with vcMMAE, mp-dLAE-MMAE and mdpr-gluc-MMAE based linkers showed high cytotoxicity to 3D spheres with efficacy similar to that in 2D cultures.

Table 11: IC50 (ng/ml) value 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 bottom clear black tissue culture treated microplates (Corning, NY) and kept adhered overnight at 37 ℃. H12F3 HGLF and non-targeted control antibodies were labeled with IncuCyte FabFluor-pH Red antibody labeling reagent (Essen Bioscience, ann Arbor, MI) according to the manufacturer's protocol. The volumes of test antibody, fabFluor reagent and medium required were calculated as 2-fold of the final assay concentration and FabFluor reagent was added in a molar ratio to antibody of 1:3. The antibody and fabfour were gently mixed and incubated at 37 ℃ for 15 minutes, and then the antibody-fabfour complex was added to each appropriate well of the cell-containing plate. The final concentration of h12F3 HGLF and unbound control antibody per well was 250ng/mL. Plates were arranged on microplate discs in incuCyte S3 (Essen Bioscience, ann Arbor, mich.) and scanned using the Adherent Cell-by-Cell protocol. Phase data and red channel data were collected (acquisition time set to 400 ms), 4 images per well, at least once every 0.5-2 hours, for up to 20 hours, with objective set to 10x. Quantification of fluorescence signal intensity was performed using an IncuCyte software analysis tool. Analysis of each cell line was refined and adjusted using label-free cell count and manual image selection for preview and algorithm training. After the analysis was completed, the data was plotted using the IncuCyte software, with the graph metric set to the mean of the red mean intensity subjects per cell normalized to the non-binding control. As shown in fig. 12, h12F3 HGLF internalized in ALPP-expressing cells, and internalization was specific, as ALPP knockout HEP2 cells did not internalize naked antibodies.

Example 6: kinetic binding and pH sensitivity

Divalent affinities were measured by biol interferometry (BLI) on an actet Red 384 system (ForteBio) with anti-human Fab-CH1 second generation (Fab 2G) biosensor at pH 7.4, 37 ℃. 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 at 3ug/mL on the biosensor for 600 seconds, then the titrated analytes hALPP and hALPP 2 were combined at 6 concentrations in the range of 0.12-125nM for 600 seconds, then the final 50 minute dissociation step was performed in kinetic buffer (1 XPBS pH 7.4 solution of 1% casein and 0.2% Tween 20). After subtracting the reference of the probe-only curve, the data were globally fitted to a 1:1 model with the Rmax sensor unconnected. The bivalent binding of h12F3 HGLF to hALPP and hALPP 2 was measured as 1.3E-10M (k) using curve fitting at concentrations of 31.3, 7.8, 1.95, 0.49 and 0.12nM, respectively _d 2.0E-05 1/s/k _a 1.5E51/Ms) and 4.4E-11M (k) _d 7.1E-06 1/s/k _a 1.6E5 1/Ms). The affinity of the HFLD variants for hALPP and hALPP 2 was reduced 26.9-fold and 34-fold, respectively, compared to HGLF, as shown in FIG. 13.

To assess pH sensitivity, divalent affinities at pH 6.0, 37 ℃ were determined using the same BLI method as the pH 7.4 experiment. The only difference was the use of different kinetic buffers (phosphate citrate solution of 1% casein and 0.2% Tween20, pH 6.0). The divalent binding of h12F3 HGLF to hALPP and hALPP 2 was measured as 6.8E-09M (k) using 800 seconds dissociation and curve fitting of concentrations 125, 31.3, 7.8, 1.95, 0.49 and 0.12nM, respectively _d 5.9E-04 1/s/k _a 8.7E4.1/Ms) and 4.8E-9M (k) _d 4.3E-04 1/s/k _a 8.9E4 1/Ms). As shown in FIG. 14, the hALPP Fc was reduced 52-fold and the hALPP 2 was reduced 109-fold compared to the pH 7.4 affinity.

Example 7: in vivo antitumor Activity

By 5X 10 ⁵ CAOV3p1 or 2.5X10 ⁶ NSG mice were inoculated subcutaneously with NCI-H1651 cells. By 1X 10 ⁷ NCI-N87, 2X 10 ⁶ Each RMUG-S, 1×10 ⁷ The LoVo and 5×10 ⁶ Subcutaneous HT-1376 cellsNude mice were vaccinated. The right flank of each mouse was inoculated subcutaneously with 0.1ml PBS and matrigel (1:1) as specified by the manufacturer. Tumor growth was monitored with calipers and the formula (0.5× [ length×width ² ]) Average tumor volumes were calculated. When the average tumor volume reaches about 100-200mm ³ At this time, mice were randomly divided into different groups, including untreated conditions or intraperitoneal injection of h12F3 HGLF or HFLD conjugated with mp-dLAE-MMAE or vcMAE, four times per four days (q 4d 4) or three times per 7 days (q 7d 3). When the tumor volume reaches about 800-1000mm ³ At this time, the mice were euthanized. % TGI is defined as (1- (mean volume of treated tumor)/(mean volume of control tumor)) ×100%. All animal procedures were conducted in institutional animal care and use committee approved protocols at the laboratory animal care assessment and certification institute.

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

The resulting tumor volumes over time for untreated mice and mice treated with 3mg/kg H12F3 ADC conjugated to vc-MMAE and dLAE-MMAE are shown in FIG. 18 for the lung tumor model NCI-H1651. ALPP ADCs exhibit similar antitumor activity in vivo. The resulting tumor growth inhibition in the seven xenograft models is shown in figure 19. The bar graph summarizes the% tumor volume change of the treated group relative to the control. The comparison was performed at 3mg/kg of h12F3-HFLD ADC conjugated to vc-MMAE and dLAE-MMAE. The average antitumor activity of the unbound ADC control is shown by the dashed line.

In another set of assays, NSG mice were inoculated subcutaneously 5X 10 ⁵ CAOV3p1, NCG mice were inoculated subcutaneously with 5X 106 SNU-2535 and nude mice were inoculated subcutaneously with 1X 10 ⁷ NCI-N87, SCID mice were inoculated subcutaneously 1X 10 ⁷ And HPACs. The right flank of each mouse was inoculated subcutaneously with 0.1ml PBS and matrigel (1:1) as specified by the manufacturer. Tumor growth was monitored with calipers and the formula (0.5× [ length×width ² ]) Average tumor volumes were calculated. When the average tumor volume reaches about 100-200mm ³ At this time, mice were randomly divided into different groups, including untreated conditions or intraperitoneal injection of h12F3HGLF conjugated with mp-dLAE-MMAE or mc-vc-MMAE, four times per 4 days (q 4 d. Times.4) or three times per 7 days (q 7 d. Times.3). When the tumor volume reaches about 2-3000mm ³ At this time, the mice were euthanized. % TGI is defined as (1- (mean volume of treated tumor)/(mean volume of control tumor)) ×100%. All animal procedures were conducted in institutional animal care and use committee approved protocols at the laboratory animal care assessment and certification institute.

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

For pancreatic tumor model HPAC, the resulting tumor volumes over time for untreated mice and mice treated with 1 or 3mg/kg h12F3 ADC conjugated to mc-vc-MMAE and mp-dLAE-MMAE are shown in FIG. 21. ALPP ADCs exhibit similar antitumor activity in vivo. The resulting tumor growth inhibition in the four xenograft models is shown in figure 22. The bar graph summarizes the percent change in tumor volume of the treated group relative to the control. The comparison was performed at 3mg/kg of h12F3-HGLF ADC conjugated with vc-MMAE and dLAE-MMAE. The average antitumor activity of the unbound ADC control is shown by the dashed line.

In another assay, twelve patient-derived xenografts were subjected to anti-tumor activity in nude mice using a 2+2 experimental design. Briefly, when enough tumor of stock animals reaches 1.0-1.5cm ³ At that time, tumors were harvested for re-implantation into animals prior to study. Animals were implanted unilaterally on the left flank with tumor fragments harvested from stock animals prior to study. Each animal was implanted from a specific passage batch and recorded. When the tumor reaches150-300mm ³ At the mean tumor volume of (c), animals were matched to the treatment or control group for dosing at the tumor volume and dosing was started on day 0. H12F3-mc-vc-MMAE conjugate was dosed at 5mg/kg (QWx 3) and compared to the PBS treated cohort. Tumor volumes were measured twice weekly. Final tumor volume measurements were made on the day the study reached endpoint. Tumor size was measured twice weekly by digital calipers starting on day 0 and data were recorded for each group, including individual and mean estimated tumor volume (mean tv±sem); tumor volumes were 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 by equation (2) using the initial (i) and final (f) tumor measurements: % tgi=1- (Tf-Ti)/(Cf-Ci). As shown in FIG. 23, the h12F3-mc-vc-MMAE conjugate SGN-ALPV showed anti-tumor activity in 58% (7/12) of the PDX models with heterologous target expression. At the doses used, the response model showed 55% to >TGI in the 100% range. Antitumor activity was observed in the PDX model from both patients receiving chemotherapy pretreatment and untreated patients (fig. 23, b and C).

Example 8: cross-reactivity and epitope mapping

To confirm the cross-reactivity of the antibodies with the ortholog ALPP protein, cynomolgus monkey (macaca falsicularis) ALPP gene (NHP ALPP) was transfected into HEK293 cells and screened for antibodies by flow cytometry. Briefly, the KD of each resulting antibody was then determined by a saturated binding assay. 1X 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 min. Cells were pelleted and washed 3 times with PBS/BSA, then 10ug/ml of APC-labeled anti-human IgG goat secondary antibody was added and incubated on ice for an additional 60 minutes. The cells were pelleted and washed 3 times with PBS/BSA and resuspended in 125. Mu.L of PBS/BSA. Fluorescence was analyzed by flow cytometry, the percentage of saturated fluorescence signal was used to determine the percent binding and then the apparent KD was calculated. Apparent K of two antibodies _D As shown in fig. 24. Heavy weight It is important that, although the antibody variant h12F3 HFLD showed significantly reduced affinity for monkey orthologous genes, HGLF variants exhibited similar binding characteristics to human and monkey placental alkaline phosphatase.

Since h12F3HGLF did not cross-react with the rat ALPP/ALPPL2 ortholog, the human ALPP region was exchanged with the rat ALPP homolog region. These constructs were transiently transfected to 2X 10 using lipofectamine 3000 (1:1.5 DNA/lipofectamine ratio) according to manufacturer's instructions ⁶ Individual cells HEK293 cells. The h12F3HGLF binding epitope was assessed on cells expressing chimeric rat/human ALPP variants 48 hours post-transfection using flow cytometry as described previously. As shown in FIG. 25, h12F3HGLF binding was impaired when the human ALPP region containing aa L287-S339 was replaced with the rat ALPP sequence.

Example 9: kinetic binding of antibodies to Fc receptors

The antibody-based immune response is driven by interactions with Fc receptors on immune cells. Thus, to determine the ability of H12F3HGLF, H12F3HGLF-mc-vc-MMAE, or H12F3HGLF-mp-dLAE-MMAE to interact with Fc receptors, the binding kinetics with hfcyri, hfcyriia H131, hfcyriia R131, hfcyriiia F158, hfcyriiia V158, and hscFcRN were assessed by Biological Layer Interferometry (BLI). Biotinylated avi-labeled human Fc receptors fused to monomeric Fc (designed and expressed in Seagen) were loaded onto a high-precision streptavidin biosensor (from ForteBio), all receptors reacted near 0.4nm, but hFc gamma R1 reacted near 1.2 nm. Initial baseline was completed in fixation buffer (0.1% BSA, 0.02% Tween 20, 1x PBS pH 7.4), then the second baseline was completed in kinetic buffer (1% casein, 0.2% Tween 20, 1x PBS pH 7.4 for hfcy RI, IIa, IIIa and IIb interactions, 1% bsa+0.2% Tween 20, phosphate citrate pH 6.0 for hscFcRN interactions). Titrated h12F3HGLF, h12F3HGLF-mc-vc-MMAE, h12F3HGLF-mp-dLAE-MMAE and positive control mAb samples were combined and dissociated as follows: in kinetic buffer, hfcyri was 600s and 1000s, hfcyriia and hfcyriib was 10s and 50s, hfcyriiia was 60s and 200s, and hsfcrn was 50s and 200s, respectively. Sensorgrams were generated on an Octet HTX system (ForteBio) at 30 ℃ and global fitting was performed with a 1:1 kinetic Langmuir isotherm model (Rmax unconnected) after subtracting the reference of the antigen loaded 0nM analyte sensor. Negative controls with highest concentration of antibody and non-immobilized Fc receptor ADC (20 μm) were also included to verify that there was no non-specific binding of the analyte to the streptavidin biosensor itself. Specific loading concentrations and times for each receptor of the streptavidin sensor and the concentration of the titrant analyte are listed (tables 12 and 13). In summary, the parent antibody and mc-vc-MMAE ADC bind all human Fc receptors, as shown in figure 26. The highest affinity for hFcγRI ranges from about 1.3 to 2.2nM, and the next highest affinity for hFcRN ranges from about 10.6 to 13.9nM. The affinity range for hfcyriia and hfcyriiia variants is 0.81-7.3 μm, while hfcyriib shows the weakest affinity range of 36-67 μm. In comparison to the positive control mAb results, the affinities of h12F3HGLF-mc-vc-MMAE and h12F3HGLF-mp-dLAE-MMAE for all human Fc receptors were very similar and comparable to the parent antibody h12F3 HGLF.

Table 12: fixed concentration and time on streptavidin biosensor

Table 11: analyte concentration

1. For 12F3 HGLF-based conjugates using mc-vc-MMAE and mp-dLAE-MMAE, the same concentration was used

Antibody Dependent Cellular Cytotoxicity (ADCC) of primary NK cells

To determine if the h12F 3HGLF backbone and derived conjugates caused Antibody Dependent Cellular Cytotoxicity (ADCC), ALPPL2 expressing cells were incubated with Natural Killer (NK) cells in vitro in the presence of h12F 3HGLF, h12F3 HGLF-mc-vc-MMAE or h12F3 HGLF-mp-dLAE-MMAE. After incubation, the percentage of cell lysis was measured. Briefly, effector cell preparation: peripheral Blood Mononuclear Cells (PBMCs) were thawed rapidly in a 37 ℃ water bath one day prior to the assay. Cells were transferred to 50mL tubes 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 1500rpm for 10 minutes. PBMC 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. Mu.g/mL and incubated at 37℃for 10 to 15 minutes. Cells were pelleted as above and resuspended in AIM-V/5% HIHS. Cells were counted and seeded in T150 flasks at a concentration of 2-4X 108 cells/flask, 25mL each. Cells were incubated undisturbed overnight at 37℃in a 5% CO2 humidified incubator. The following day, non-adherent cells were collected and the flask was vigorously rinsed 3 times (7 mL) with PBS. The wash was combined with non-adherent cells and pelleted by centrifugation at 1500rpm for 7 minutes. Cells were resuspended in a small volume (2 mL) for counting, and the cell suspension was adjusted to a concentration of 5×107 cells/mL in pbs+2% fbs (recommended according to EasySep protocol). NK cells were isolated by negative selection according to the EasySep Human NK cell enrichment kit (Stem cell technology, catalog No. 19055) instructions. The enriched effector cells were then suspended in RPMI/1% FBS at a concentration of 7.2×105 cells/mL (such that 70 μl contains about 5×104 effector cells). Preparation of target cells: loVo cells expressing ALPPL2 were collected and counted. Next, 5X 106 cells were removed and pelleted by centrifugation. Cells were resuspended in 100 μl of FBS. Then, 100 μl (about 100 μci) of Cr-51 (Perkin Elmer Hea lth Sciences, inc., catalog No. NEZ 030S) was added to the cells and mixed by tapping. Cells were placed in 37 ℃, 5% CO2, humidified incubator for 1 hour, and occasionally tapped into tubes to suspend the cells. Cells were washed 3 times with RPMI/1% FBS. The tube was tapped between washes to loosen the cell pellet. After washing, cells were resuspended in 10mL RPMI/1% FBS and counted. Then, 7.2X105 cells were removed and suspended in a total volume of 10mL of the assay medium so that 70. Mu.L corresponded to-5X 103 target cells. Preparation of ADC and antibody dilutions and plate assembly: antibodies and ADCs were diluted at 3x concentration in assay medium. The antibodies tested were h12F 3HGLF, h12F3 HGLF-mc-vc-MMAE or h12F3 HGLF-mp-dLAE-MMAE, CD71 binding afucosylated antibodies and isotype control. Antibodies were added to the assay plates prior to the addition of Cr-labeled target cells. In addition, 70 μl and 140 μl of assay medium were added in place of the antibody to control wells representing total release control and spontaneous release control, respectively. Finally, the target cells were mixed and 70 μl was added to each of the test and control wells of the 96-well plate. The targets were incubated with mAb for 30 min at 37℃in a 5% CO2 humidified incubator. Then, 70. Mu.L (5X 104) effector cells were added to each test well, while 70. Mu.L of 3% Triton X-100 was added to the total release well and mixed. The plates were returned to 37 ℃, 5%, CO2, and humidified in an incubator for 4 hours. After incubation, 35 μl of supernatant was transferred to a Luma plate. The Luma plates were dried overnight, then covered with sealing tape and read on a Perkin Elmer TopCount NXT microplate scintillation counter. Analysis was performed by calculating% specific lysis (analyzed with GraphPad Prism) as follows: % specific lysis = [ (test cpm-background cpm)/(total cpm-background cpm) ]. Times.100. NK cytotoxicity was mediated in vitro in the presence of the h12F 3HGLF antibody as well as h12F3 HGLF-mc-vc-MMAE and h12F3 HGLF-mp-dLAE-MMAE, as shown in FIG. 27. This activity is similar to the positive control and is mediated by the presence of targets on the cells, since non-binding antibodies are unable to stimulate effector cells.

Antibody dependent cytotoxicity

To determine whether h12F3HGLF, h12F3HGLF-mc-vc-MMAE or h12F3HGLF-mp-dLAE-MMAE showed ADCP activity, ALPP/ALPPL2 expressing antibodies or ADC coated fluorescent cells were co-incubated with primary macrophages and phagocytosis measured by fluorescence flow cytometry. Briefly, loVo tumor cells were fluorescently labeled with PKH26 according to the manufacturer's instructions. Cells were harvested from dishes with 0.05% trypsin EDTA and washed once with 1 xPBS. Cells were resuspended in 1mL of diluent C contained in PKH26 red fluorescent cell membrane labelling kit (Sigma-Aldrich, catalog number PKH26GL-1 KT). In a separate tube, 1mL of diluent c+4 μl PKH26 dye is added and pipetted up and down for mixing. The staining solution was transferred to resuspended cells and mixed rapidly by pipetting up and down multiple times. The cells were incubated at room temperature for 5 minutes and the labelling reaction was terminated by the addition of 2mL FBS. Cells were washed 3 times with RPMI/10% FBS and resuspended in PBS at a concentration of 0.8×106 cells/mL. 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 procedure. In a separate 96-well U-plate, 10-fold mAb, ADC and stock of isotype control in PBS were serially diluted 1:10 in PBS and 33. Mu.L/well was added to the appropriate wells of cells in the U-plate. Plates were incubated for 30 min at room temperature, centrifuged, and washed once with 200. Mu.L/Kong Peiyang base (RPMI/10% FBS). Cells were resuspended in 330. Mu.L/Kong Peiyang base (RPMI/10% FBS). On the day before the assay, PBMC from 2 healthy donors were thawed in a 37℃water bath and transferred to RPMI/10% FBS (0.1-0.2 EU/mL). A total of 0.7×106 PBMCs per well were added to a 48 well flat bottom plate and allowed to adhere overnight. Old medium (and non-adherent cells) was aspirated and replaced with 200 μl fresh medium. Next, 100. Mu.L of labeled, treated target cells in each well were transferred in triplicate to the corresponding wells of the adherent monocyte/macrophage planar bottom plate, and the plates were incubated overnight at 37℃for 16-18 hours. All cells in the 48-well plate were harvested by collecting the loading, collecting the washing with 1x PBS and isolating with 1x Versene. Macrophages were fluorescently labeled using the following steps: target cells and macrophages were collected in U-shaped plates, centrifuged, resuspended in 50. Mu. LFACS staining buffer containing human Fc fragment blocking agent (1:20 dilution) and incubated on ice for 30 min. Next, 50. Mu.L of a 1:50 dilution of CD14-BV421 and CD45-APC-Cy7 antibodies diluted in FACS staining buffer was added to each well and incubated in foil for 30 minutes on ice. The cells were centrifuged, washed 2 times with FACS buffer and resuspended in 1x PBS for subsequent flow cytometry analysis on an Attune NxT flow cytometer. YL1 GeoMean fluorescence of CD14+/CD45 cells (CD14+/CD45+ cells MFI) was analyzed using FlowJo, and values were then exported to Excel and GraphPad Prism for further data analysis. Phagocytosis is reported as MFI of cd14+ cells. As shown in fig. 28, the presence of h12F3HGLF, h12F3HGLF-mc-vc-MMAE or h12F3HGLF-mp-dLAE-MMAE conjugates enabled phagocytosis of target expressing cells with similar kinetics as the positive control (anti-CD 47 antibody). This activity is dependent on the presence of ALPPL2 expression on the target cells, as non-binding antibodies do not cause any cell death.

In another assay, fcyriii-dependent antibody-dependent cytotoxicity (ADCC) was measured by using a surrogate luciferase-mediated based bioassay by Promega. Briefly, ALPP/ALPPL2 expressing cells were plated on 96-well plates and co-cultured with ADCC bioassay effector cells (Promega) in the presence of increased amounts of naked h12F3 antibody or h12F3HGLF antibody 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 an Envision platform. As shown in fig. 29, h12F3HGLF was able to activate fcgrii signaling in the reporter cell line with similar kinetics as the h12F3HGLF ADC conjugated mc-vc-PABC-MMAE. Conjugation to 8 MDpr-PEG (12) -gluc-MMAE molecules reduced ADCC activity compared to naked h12F3 HGLF.

Example 10: immunogenic cell death

Signaling pathway activation for immunogenic cell death

To determine if h12F3 HGLF-mc-vc-MMAE and h12F3 HGLF-mp-dLAE-MMAE could activate ICD markers, ALPP/ALPPL2 expressing cells were treated with ADC and immunoblotted to determine the phosphorylation status of IRE and JNK pathways. Briefly, four million LOVO cells were plated in 10mL of competitive growth media (Ham's F-12K (Kaighn's) media+10% FBS) in 10cm TC-treated dishes and allowed to adhere overnight. Cells were treated with 10nM MMAE or 1 and 10ug/mL 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 and resuspended in 500uL cold PBS and transferred to Eppendorf tubes. The sample was rotated at 300Xg for 3 minutes at 4 degrees. The supernatant was removed and the cells resuspended in RIPA lysis buffer containing protease and phosphatase. After incubation on ice for 5 minutes, the samples were spun at 17000Xg for 10 minutes at 4 degrees, the supernatant collected and stored at-80 ℃. Quantitative samples were separated in NuPAGE 4% -12% bis-tris gels and run with smaller proteins in MES running buffer or larger proteins in MOPS (165 v 40 min). The gel was transferred to PVDF membrane using iBlot2 (20 v,7 min). The membranes were briefly rinsed in DI water and then placed in blocking buffer (TBS+0.1% tween-20+5% BSA) overnight at 4 ℃. The blots were then incubated with primary antibodies to IRE, JNK, p-IRE or p-JNK at 1:1000 dilution in blocking buffer for 2 hours at room temperature. 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 antibodies were prepared at 1:10,000 dilutions in blocking buffer. The blots were incubated with secondary antibody for 1 hour at room temperature. The blots were again washed 3 times with TBST. Blot analysis was performed using SignalFire ECL and imaged on an Amersham imager 600. The blots were then stripped and GAPDH was re-probed as a loading control and blotted as described above. As shown in FIG. 30, incubation of LoVo cells with h12F3 HGLF-mc-vc-MMAE or h12F3 HGLF-mp-dLAE-MMAE increased the phosphorylation levels of pIRE and pJNK, which play a key role in the activation of the immunogenic cell death process.

To determine if treatment with h12F3 HGLF ADC primer resulted in ATP release in the medium, 600,000 LOVO cells were plated in 2mL of competitive growth medium (Ham's F-12K (Kaighn's) medium +10% FBS) in 6-well TC-treated dishes and allowed to adhere overnight. Solutions of 10nM MMAE,1 or 10. Mu.g/mL h12F3 HGLF mp-dLAE-MMAE or mc-vc-MMAE were prepared in complete medium. The cells were then incubated in the treated medium for 24, 48 or 72 hours. At each endpoint, 500uL of supernatant was collected from each well (sample). The supernatant was spun at 200Xg, 4 degrees for 1 minute to carefully remove any cell debris. Then 3 50uL aliquots of each supernatant (for triplicate data) were placed in a black-wall, transparent-bottom 96-well plate. 50uL of reconstituted Cell Titer Glo was then added to all wells containing supernatant. The plate was covered and protected from light. The plate was then read on an Envision microplate reader. Raw luminescence data from triplicate data for all samples were averaged. To determine the fold change from untreated samples, the average luminescence of the experimental samples was divided by the average of untreated samples. As shown in FIG. 31, both h12F3 HGLF mp-dLAE-MMAE or mc-vc-MMAE conjugates resulted in the release of ATP, which is a marker of immunogenic cell death.

Example 11: pharmacokinetics of

Pharmacokinetic assessments of humanized h12F3 ADCs were performed in non-human primates. Antibody drug conjugates comprising h12F3 HGLF-vc-MMAE (4) and HGLF-dLAE-MMAE (4) were administered once at 1mg/kg and plasma samples were collected at the indicated 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 from dosing samples diluted in pooled cynomolgus monkey K2EDTA plasma (BioIVT). And diluting the research sample with the concentration of the test sample exceeding the determination limit to a certain range by using the K2EDTA plasma of the cynomolgus monkey which is not contacted with the medicine. Standards, QC and study samples were diluted to a Minimum Required Dilution (MRD) of 1:20 in Rexxip HX buffer (Gyros Protein Technologies, sweden). A30 nM equimolar master mix solution was prepared by diluting biotinylated anti-human kappa light chain (Seagen) and Ale xaFlour-647 anti-human Fcγ (Jackson Immunoresearch) in a 1 Xphosphate buffered saline solution containing 0.01% (v/v) tween-20 (PBST). Equal volumes of MRD standard, QC or study sample and master mix solution 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 within the CD. The column was washed 4 times with PBST and the relevant fluorescence on the column was detected at 635 nm. Fluorescence response of the calibrator was fitted to 5-parameter logistic regression (5-PL) using Gyrolab Evaluator software. Total h12F3 HGLF-vcMMAE and HGLF-dLAE-MMAE QC and study sample concentrations were interpolated according to respective fitted standard curves and used for pharmacokinetic assessment. PK parameters were determined by non-atrioventricular analysis (NCA) using Phoenix WinNonlin (version 8.2, certara USA, inc.) as appropriate. The following PK parameters were determined: area under the plasma concentration-time curve (AUC 0-21) up 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 linearity method. Half-life, cl and Vss are reported for plasma concentration-time curves that regulate R2. Gtoreq.0.8 and extrapolate AUC0-inf < 20%. The resulting pharmacokinetic parameters are shown in table 14, showing that h12F3 HG LF conjugates with both vcMMAE and mp-dLAE-MMAE show similar antibody conjugated MMAE pharmacokinetic profiles with no evidence of target-mediated drug treatment when compared to non-binding ADC controls.

TABLE 14

To quantify antibody conjugated MMAE (acMMAE), plasma samples were first immunocaptured at 2-8 ℃ to isolate ADC (GE Healthcare) for one hour. The bound samples were washed with papain digestion buffer (20 mM KPO4, 10mM EDTA, 20mM cysteine HCl) and then 2mg/mL papain in digestion buffer was added to each sample. The samples were incubated at 37℃for four hours to enzymatically release acMMAE. The resulting released acMMAE was extracted using solid phase extraction. Each sample was then analyzed using normal phase UPLC (Betasil, thermosipher) in combination with tandem mass spectrometry (Sciex 6500+triple Quad). Table 15 shows similar pharmacokinetic parameters for antibody conjugated MMAE using h12F3 HGLF vc-MMAE and HGLF-dLAE-MMAE conjugates, the latter showing an extended half-life.

TABLE 15

Tolerance of h12F3 HGLF ADC was assessed in cynomolgus monkeys, a pharmacologically relevant species, with comparable binding affinities to human and cynomolgus ALPP orthologs. Female monkeys were administered 5mg/kg of h12F3 HGLF-vc-MMAE (4) or 5, 8, 9, and 10mg/kg of h12F3 HGLF-dLAE-MMAE (4), respectively, once a week for four weeks (q 1wx 4). Toxicology assessments include body weight, clinical observations, hematology, coagulation, serum chemistry, and TK. At necropsy at the end-stage (1 week after the last dose) and recovery period (4 weeks after the last dose), gross pathology examination was performed and histopathology examination was performed on the tissues. The maximum tolerated dose for h12F3 HGLF-vc-MMAE (4) was 5mg/kg, and the maximum tolerated dose for h12F3 HGLF-dLAE-MMAE (4) was 9mg/kg (Table 16). Both ADCs were tested for MMAE pharmacological consistent myelotoxicity by hematological and anatomical pathology assessment, and dose-limiting toxicity was considered. Pulmonary alveolar macrophage accumulation, reduced secondary and tertiary follicular numbers in the ovaries, and other toxicities of lymphoid depletion in thymus

Table 16

Incorporated by reference

All references cited herein, including patents, patent applications, scientific articles, textbooks, and the like, are hereby incorporated by reference in their entirety.

Sequence listing

<110> Si into stock Co., ltd

<120> anti-ALPP/ALPPL 2 antibody and antibody-drug conjugate

<130> 5620-00112PC

<150> US 63/162,635

<151> 2021-03-18

<150> US 63/301,574

<151> 2022-01-21

<160> 74

<170> PatentIn version 3.5

<210> 1

<211> 1608

<212> DNA

<213> Homo sapiens (Homo sapiens)

<400> 1

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 cagtgggagt 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 acccccgcgg 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 (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 (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

tcagtgggag 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 (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 (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 (Artificial Sequence)

<220>

<223> mu IGHV 7-3.04-closest murine 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 (Artificial Sequence)

<220>

<223> hu IGHV3-49.01/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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (Artificial Sequence)

<220>

<223> mu IGKV 19-93.01-closest murine 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 (Artificial Sequence)

<220>

<223> hu IGKV1-33.01/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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (Artificial Sequence)

<220>

<223> h12F 3L 1 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 (Artificial Sequence)

<220>

<223> h12F 3L 2 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 (Artificial Sequence)

<220>

<223> h12F 3L 3 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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (Artificial Sequence)

<220>

<223> LD, LF CDR 2, IMGT

<400> 69

Tyr Thr Ser

1

<210> 70

<211> 8

<212> PRT

<213> Artificial sequence (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 (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 (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 (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 (Artificial Sequence)

<220>

<223> h12F3 epitope-ALPPL 2

<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;

CDR-L2 comprising the amino acid sequence of SEQ ID NO. 64 or SEQ ID NO. 69; a kind of electronic device with high-pressure air-conditioning system

CDR-L3 comprising the amino acid sequence of SEQ ID NO. 65 or SEQ ID NO. 70;

wherein the CDRs are 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 comprising the amino acid sequence of SEQ ID NO. 58; CDR-L1 comprising the amino acid sequence of SEQ ID NO. 63; CDR-L2 comprising the amino acid sequence of SEQ ID NO. 64; and CDR-L3 comprising the amino acid sequence of SEQ ID NO. 65; wherein the 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; CDR-H3 comprising the amino acid sequence of SEQ ID NO. 62; CDR-L1 comprising the amino acid sequence of SEQ ID NO. 68; CDR-L2 comprising the amino acid sequence of SEQ ID NO. 69; and CDR-L3 comprising the amino acid sequence of SEQ ID NO. 70; wherein the CDR is determined by IMGT.

4. The antigen binding protein or fragment of any one of claims 1-3, comprising a VH and a VL, wherein the VH has at least 80%, 85%, 90%, 95% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID No. 15, and wherein the VL has at least 80%, 85%, 90%, 95% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID No. 30.

5. The antigen-binding protein or fragment of any one of claims 1-4, comprising a VH and a 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, comprising 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, comprising a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID No. 15, and the VL comprises the amino acid sequence of SEQ ID No. 30.

8. The antigen binding protein or fragment of any one of claims 1-7, comprising: 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: HC comprising the amino acid sequence of SEQ ID NO. 40 and comprising: 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 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 or cytostatic agent.

15. The antibody-drug conjugate of claim 14, wherein the antibody or antigen binding fragment is conjugated to the cytotoxic or cytostatic agent through a linker.

16. The antibody-drug conjugate of any one of claims 14-15, wherein the cytotoxic 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 through 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 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 being 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 a p-aminobenzyl alcohol (PABC) group having the structure of formula II;

22. the antibody-drug conjugate of any one of claims 14-21, wherein the linker is attached to monomethyl auristatin E, thereby forming an antibody-drug conjugate having the structure:

wherein 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;

subscript nn is a number from 1 to 5;

subscript a 'is 0 and a' is absent;

p1, P2 and P3 are each amino acids, wherein:

the first of the amino acids P1, P2 or P3 is negatively charged;

a second one of the amino acids P1, P2 or P3 has an aliphatic side chain with a hydrophobicity not greater than leucine; and is also provided with

The third of the amino acids P1, P2 or P3 is less hydrophobic than leucine,

wherein the first one of the amino acids P1, P2 or P3 corresponds to any one of P1, P2 or P3, the second one of the amino acids P1, P2 or P3 corresponds to one of the two remaining amino acids P1, P2 or P3, and the third one of the amino acids P1, P2 or P3 corresponds to the last remaining amino acid P1, P2 or P3,

provided 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 is in a D-amino acid configuration;

one of the P2 and P1 amino acids has an aliphatic side chain with lower hydrophobicity than leucine; and is also provided with

The other 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 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-Asp-or-D-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,

wherein 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 that produces the antigen binding protein or fragment of any one of claims 1-13.

37. A method of making an antigen binding protein or fragment thereof, comprising culturing the host cell of claim 36 under conditions suitable for production of 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 ALPP and/or ALPPL2 expressing cancer in a subject, comprising administering to a subject in need thereof an effective amount of the antigen binding protein or fragment of any one of claims 1-31 or 40, or the pharmaceutical composition of 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/ALPPL 2 antibody conjugated to mc-vc-PABC-MMAE, wherein the antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:30, and wherein the antibody-drug conjugate has the structure:

wherein Ab is the antibody and p represents a number from 1 to 16.

46. An antibody-drug conjugate comprising an antigen binding protein or fragment thereof that binds ALPP and ALPPL2, wherein the antibody-drug conjugate is represented by the structure:

or a pharmaceutically acceptable salt thereof, wherein:

ab is an anti-ALPP/ALPPL 2 antibody and p represents a number from 1 to 12;

subscript nn is a number from 1 to 5;

subscript a 'is 0 and a' is absent;

p1, P2 and P3 are each amino acids, wherein:

the first of the amino acids P1, P2 or P3 is negatively charged;

a second one of the amino acids P1, P2 or P3 has an aliphatic side chain with a hydrophobicity not greater than leucine; and is also provided with

The third of the amino acids P1, P2 or P3 is less hydrophobic than leucine,

wherein the first one of the amino acids P1, P2 or P3 corresponds to any one of P1, P2 or P3, the second one of the amino acids P1, P2 or P3 corresponds to one of the two remaining amino acids P1, P2 or P3, and the third one of the amino acids P1, P2 or P3 corresponds to the last remaining amino acid P1, P2 or P3,

provided 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 is in a D-amino acid configuration;

one of the P2 and P1 amino acids has an aliphatic side chain with lower hydrophobicity than leucine; and is also provided with

The other 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 the P3 amino acid is D-Leu or D-Ala, the P2 amino acid is Ala, glu, or Asp, and the P1 amino acid is Ala, glu, or Asp.

51. The antibody-drug conjugate of any of claims 46-50, wherein-P3-P2-P1-is-D-Leu-Ala-Asp-, -D-Leu-Ala-Glu-, -D-Ala-Asp-or-D-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,

wherein Ab is an anti-ALPP/ALPPL 2 antibody and p represents a number from 1 to 12.

54. An antibody-drug conjugate comprising an isolated anti-ALPP/ALPPL 2 antibody conjugated to mp-dLAE-PABC-MMAE, wherein the antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:30, and wherein the antibody-drug conjugate has the structure:

wherein 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, said antigen binding protein or fragment thereof being capable of binding one or more amino acids of a peptide comprising SEQ ID No. 73 and/or SEQ ID No. 74.