HK1181402B - Anti-fap antibodies and methods of use - Google Patents

Description

Anti-fibroblast activation protein antibodies and methods of use

Technical Field

The present invention relates to antibodies specific for Fibroblast Activation Protein (FAP). In addition, the invention relates to polynucleotides encoding such antibodies, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the antibodies and methods of using them in the treatment of disease.

Background

Fibroblast Activation Protein (FAP) and anti-FAP antibodies

Human fibroblast activation protein (FAP; GenBank accession AAC51668), also known as Seprase, is a 170kDa membrane-integrated serine peptidase (EC3.4.21. B28). FAP belongs to the family of dipeptidyl peptidases IV (Yuetal., FEBSJ277,1126-1144(2010)), together with dipeptidyl peptidase IV (also known as CD 26; GenBank accession number P27487), a closely related cell surface enzyme, and other peptidases. It is a homodimer, containing two N-glycosylated subunits with a larger C-terminal extracellular domain in which the catalytic domain of the enzyme is located (scanlane et al, proc natl acad sciusa91,5657-5661 (1994)). FAP has both prolyl-post-dipeptidyl peptidase and gelatinase activities in its glycosylated form (suneal, protein exprppurif24, 274-281 (2002)).

Human FAP was originally identified in cultured fibroblasts using monoclonal antibody (mAb) F19 (described in WO93/05804, ATCC No. HB 8269). Homologs of the protein are found in a variety of species, including mice (Niedermeyewet et al, IntJcancer71,383-389(1997); Niedermeyewet et al, EurJbiochem254,650-654(1998); GenBank accession No. AAH 19190). FAP has a unique organizational profile: its expression was found to be highly upregulated on reactive stromal fibroblasts in more than 90% of all primary and metastatic epithelial tumors, including lung, colorectal, bladder, ovarian and breast carcinomas, but it is not normally present in normal adult tissues (rettigtal, proc natl acad sciusa85,3110-3114(1988), Garin-chesae, proc natl acad sciusa87,7235-7239 (1990)). Subsequent reports have shown that FAP is expressed not only in stromal fibroblasts but also in some malignant cell types of epithelial origin, and that FAP expression is directly linked to the malignant phenotype (jinetal, anticancer res23,3195-3198 (2003)).

Due to its expression in a variety of common cancers and its limited expression in normal tissues, FAP is considered promising as an antigenic target for imaging, diagnosis and therapy of a variety of carcinomas. Thus, a variety of monoclonal antibodies have been generated against FAP for research, diagnostic and therapeutic purposes.

Celuzumab (Sibrotuzumab)/BIBH1, a humanized version of the F19 antibody that specifically binds to human FAP (described in WO 99/57151), and another humanized or fully human antibody specific for the FAP antigen with an F19 epitope (described in Mersmann et al, IntJCancer92,240-248(2001); Schmidtetal, EurJBiochem268,1730-1738(2001); WO 01/68708) were developed. The OS4 antibody is another humanized (CDR grafted) version of the F19 antibody (wuestetal., jbiotech92,159-168(2001)), whereas scFv33 and scFv36 have different binding specificities than F19 and are cross-reactive for human and mouse FAP proteins (brockset al, molmed7,461-469 (2001)). More recently, other murine anti-FAP antibodies, as well as chimeric and humanized forms thereof, have been developed (WO2007/077173, ostrermanentanal, clinencearres 14,4584-4592 (2008)).

Proteases in the tumor stroma drive processes such as angiogenesis and/or tumor cell migration through proteolytic degradation of extracellular matrix (ECM) components. Furthermore, tumor stroma plays an important role in the supply of nutrients and oxygen to the tumor, as well as in tumor invasion and metastasis. These basic functions make it a potential therapeutic target as well as a diagnostic target.

In one item use¹³¹Evidence of the feasibility of the concept of targeting tumor stroma in vivo using an anti-FAP antibody was obtained in phase I clinical studies with I-labeled F19 antibody, demonstrating the specific enrichment of this antibody in tumors and the detection of metastasis (weltal, jinoncocol 12,1193-1203 (1994)). Similarly, a phase I study with sirolimumab demonstrated¹³¹I specific tumor accumulation of labeled antibody (scottet., clincancer res9,1639-1647 (2003)). However, one early phase II trial of unconjugated sirolimus in patients with metastatic colorectal cancer was discontinued due to the lack of efficacy of the antibody in inhibiting tumor development (hofheinzetal, Onkologie26,44-48 (2003)). Also, a more recently developed anti-FAP antibody failed to show an in vivo anti-tumor effect in unconjugated form (WO 2007/077173).

Thus, there remains a need for enhanced therapeutic approaches, including antibodies with improved efficacy targeting FAP, to treat cancer.

Antibody glycosylation

The oligosaccharide component can significantly affect properties related to the efficacy of the therapeutic glycoprotein, including physical stability, resistance to protease attack, interaction with the immune system, pharmacokinetics, and specific biological activity. Such properties may depend not only on the presence or absence of oligosaccharides, but also on the specific structure of the oligosaccharides. Some generalizations between oligosaccharide structure and glycoprotein function can be made. For example, certain oligosaccharide structures mediate the rapid clearance of glycoproteins from the bloodstream via interaction with specific carbohydrate-binding proteins, while other oligosaccharide structures may be bound by antibodies and trigger unwanted immune responses. (Jenkinoset al, Nature Biotechnol14,975-81 (1996)).

IgG 1-type antibodies (i.e., the most commonly used antibodies in cancer immunotherapy) are glycoproteins with conserved N-linked glycosylation sites at Asn297 in each CH2 domain. Two complex biantennary oligosaccharides attached to Asn297 are buried between each CH2 domain, form extensive contacts with the polypeptide backbone, and their presence is critical for antibody-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) (lifelyet al, glycobiology5,813-822(1995); jefferiset al, immunol rev163,59-76(1998); wrightand morrison, trends biotechnol15,26-32 (1997)). Protein engineering studies have shown that Fc γ R interacts with the lower hinge region of IgGCH2 domains. Lundetal, J.Immunol.157:4963-69 (1996). However, Fc γ R binding also requires the presence of oligosaccharides in the CH2 region. Lundetal, J.Immunol.157:4963-69(1996); WrightandMorrison, trends Biotech.15:26-31(1997) suggest that either oligosaccharides and polypeptides directly contribute to the interaction site or oligosaccharides are required for maintaining the conformation of an active CH2 polypeptide. Therefore, modification of oligosaccharide structure can be explored as a means for improving the interaction affinity between IgG1 and Fc γ R and improving ADCC activity of IgG 1.

One way to achieve a substantial increase in the potency of monoclonal antibodies is to enhance their natural, cell-mediated effector functions by engineering their oligosaccharide components as described inet al, NatBiotechnol17,176-180(1999) and U.S. Pat. No.6,602,684(WO99/54342), the contents of which are incorporated herein by reference in their entirety.Et al show that overexpression of β (1,4) -N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase that catalyzes the formation of two bisected oligosaccharides, in Chinese Hamster Ovary (CHO) cells significantly increases the in vitro ADCC activity of antibodies produced in those cellsType (la) antibodies show strongly enhanced ADCC, but only antibodies in which most of the N-glycans are of the complex type are capable of inducing significant complement dependent cytotoxicity (ferraraet al, biotechn bioengg 93,851-861 (2006)). Compositional changes in Asn297 carbohydrate or elimination thereof also affect the binding of the antibody Fc domain to the Fc γ receptor (Fc γ R) and complement C1q proteins, which are important for ADCC and CDC, respectivelyetal.,NatBiotechnol17,176-180(1999);Daviesetal.,BiotechnolBioeng74,288-294(2001);Mimuraetal.,JBiolChem276,45539-45547(2001);Radaevetal.,JBiolChem276,16478-16483(2001);Shieldsetal.,JBiolChem276,6591-6604(2001);Shieldsetal.,JBiolChem277,26733-26740(2002);Simmonsetal.,JImmunolMethods263,133-147(2002))。

Summary of The Invention

The present invention provides antibodies that specifically bind to Fibroblast Activation Protein (FAP), have high affinity, and/or enhanced effector function.

In one aspect, the invention relates to an antibody that specifically binds FAP, comprising at least one (i.e., one, two, three, four, five, or six) of the complementary regions (CDRs) set forth in seq id nos3, 5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175, and 177. In one embodiment, the antibody comprises three heavy chain CDRs selected from the group consisting of seq id nos3, 5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175, and 177 (i.e., HCDR1, HCDR2, and HCDR 3)And/or three light chain CDRs (i.e., LCDR1, LCDR2, and LCDR 3). In a more particular embodiment, the antibody comprises an antibody heavy chain variable region and/or an antibody light chain variable region, particularly both heavy and light chain variable regions, selected from the heavy and light chain variable region sequences of seq id nos 193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309 and 311. In one embodiment, the antibody comprises an Fc region, particularly an IgGFc region. In yet another embodiment, the antibody is a full length antibody, particularly an IgG class antibody. In another embodiment, the antibody comprises a human antibody constant region. In one embodiment, the antibody is human. In one embodiment, the antibody is engineered by glycoengineering to have a modified oligosaccharide in the Fc region. In one embodiment, the antibody has an increased proportion of nonfucosylated and/or bisected oligosaccharides in the Fc region as compared to the nonglycoengineered antibody. In yet another embodiment, the antibody has increased effector function and/or increased Fc receptor binding affinity. In a particular embodiment, the increased effector function is increased antibody-dependent cell-mediated cytotoxicity (ADCC). In another embodiment, the antibody has a K of less than about 1 μ M, preferably less than about 100nM, most preferably less than about 1nM or even less than about 0.1nM_DThe values are combined with FAP. In one embodiment, the antibody is affinity matured. In one embodiment, the antibody binds FAP in human tissue. In one embodiment, the antibody does not induce FAP internalization.

In other aspects, the invention also relates to polypeptides, polynucleotides, host cells, and expression vectors related to the antibodies. In yet another aspect, the invention relates to a method of making the antibody. In yet another aspect, the invention relates to methods of using the antibodies, particularly for treating diseases characterized by FAP expression (such as cancer).

Brief Description of Drawings

Figure 1 shows a Surface Plasmon Resonance (SPR) -based kinetic analysis of affinity matured anti-fafab fragments. Processed kinetic datasets are presented for clone 19G1 binding to human (hu) fap (a) and murine (mu) fap (B), for clone 20G8 binding to hufap (c), mufap (d), and for clone 4B9 binding to hufap (e) and mufap (f). The smooth line presents the overall fit of the data to the 1:1 interaction model.

Figure 2 shows SPR-based kinetic analysis of affinity matured anti-fafab fragments. Processed kinetic data sets were presented for clone 5B8 binding to hufap (a) and mufap (B), for clone 5F1 binding to hufap (c), mufap (d), and for clone 14B3 binding to hufap (e) and mufap (F). The smooth line presents the overall fit of the data to the 1:1 interaction model.

Figure 3 shows SPR-based kinetic analysis of affinity matured anti-fafab fragments. Processed kinetic data sets were presented for clone 16F1 binding to hufap (a) and mufap (b), for clone 16F8 binding to hufap (C), mufap (d), and for clone O3C9 binding to hufap (e) and mufap (F). The smooth line presents the overall fit of the data to the 1:1 interaction model.

Figure 4 shows SPR-based kinetic analysis of affinity matured anti-fafab fragments. Processed kinetic data sets were presented for clone O2D7 binding to hufap (a) and mufap (b), for clone 28H1 binding to hufap (c), mufap (D), cynomolgus monkey (cyno) fap (e), and for clone 22A3 binding to hufap (f), mufap (g) and cynofap (H). The smooth line presents the overall fit of the data to the 1:1 interaction model.

Figure 5 shows SPR-based kinetic analysis of affinity matured anti-fafab fragments. Processed kinetic data sets were presented for clone 29B11 in combination with hufap (a), mufap (B), cynofap (C) and for clone 23C10 in combination with hufap (d), mufap (e) and cynofap (f). The smooth line presents the overall fit of the data to the 1:1 interaction model.

Figure 6 shows SPR-based kinetic analysis of 3F2(a), 4G8(B) and 3D9(C) anti-FAP antibodies (as Fab fragments) binding to human, mouse and cynomolgus FAP. The processed kinetic data set is presented, and the smooth line presents the overall fit of the data to the 1:1 interaction model.

Figure 7 shows SPR-based kinetic analysis of 3F2(a), 4G8(B) and 3D9(C) anti-FAP antibodies (as human IgG) binding to human, mouse and cynomolgus FAP. The processed kinetic data set is presented, and the smooth line presents the overall fit of the data to the 1:1 interaction model.

Fig. 8 shows representative pictures of human samples of (a) non-small cell lung cancer (NSCLC) immunohistochemically stained for FAP using 2F3 mouse IgG2a antibody, (B) colon adenocarcinoma immunohistochemically stained for FAP using 2F3 mouse IgG2a antibody, (C) colon adenocarcinoma immunohistochemically stained for FAP using 3D9 mouse IgG2a antibody, and (D) colon adenocarcinoma immunohistochemically stained for FAP using 4G8 mouse IgG2a antibody. FAP was detected by all antibodies and in the tumor stroma in all samples (left panel), while no staining was observed for isotype control antibodies (right panel).

Figure 9 shows binding of human IgG1 anti-FAP antibody to FAP expressed on HEK293 cells stably transfected with human (a) or murine (B) FAP, as determined by FACS.

Figure 10 shows binding of human IgG1 anti-FAP antibody to DPPIV (CD26) or HER2 expressed on stably transfected HEK293 cells, as determined by FACS. Trastuzumab (trastuzumab) as an anti-HER 2 antibody and anti-CD 26 antibody were used as positive controls. As negative controls, secondary antibodies, control IgG or no antibody at all (cells only) were used.

Figure 11 shows binding of human IgG1 anti-FAP antibody to FAP on human fibroblasts (cell line GM 05389) as determined by FACS. Secondary antibodies or no antibody at all was used as negative controls.

Figure 12 shows binding of human IgG1 anti-FAP antibody to human fibroblasts (cell line GM 05389), different human tumor cell lines, or HEK293 cells stably transfected with human FAP, as determined by FACS.

Fig. 13(a) and (B) show FAP expression levels on GM05389 lung fibroblast cell surface at different time points after incubation with anti-FAP human IgG1 antibody 3F2 or 4G8, as determined by FACS. No significant reduction in FAP expression levels was observed, indicating FAP internalization. Secondary antibody alone was shown as a negative control.

Figure 14 presents representative immunofluorescence images showing plasma membrane staining on GM05389 lung fibroblasts obtained after 4 ℃ 45 min (a), 37 ℃ 20 min (B), 37 ℃ 1 hr (C), or 37 ℃ 6 hr (D) anti-FAP 4G8IgG binding. The anti-CD 20 antibody GA101 used as isotype control showed background staining. EEA1 marks the early endosome. Note that FAP surface plasma membrane staining lasts up to 6 hours after binding of anti-FAP 4G8 antibody.

FIG. 15 shows the purification and analysis of wild-type 28H1 human IgG. A) Protein A affinity chromatography purification step. B) A size exclusion chromatography purification step. C) Analytical SDS-PAGE. D) Analytical size exclusion chromatography. The experimental protocol was as described in example 1.

Figure 16 shows purification and analysis of glycoengineered 28H1 human IgG. A) Protein A affinity chromatography purification step. B) A size exclusion chromatography purification step. C) Analytical SDS-PAGE. D) Analytical size exclusion chromatography. The experimental protocol was as described in example 1.

FIG. 17 shows the purification and analysis of wild-type 29B11 human IgG. A) Protein A affinity chromatography purification step. B) A size exclusion chromatography purification step. C) Analytical SDS-PAGE. D) Analytical size exclusion chromatography. The experimental protocol was as described in example 1.

Figure 18 shows purification and analysis of glycoengineered 29B11 human IgG. A) Protein A affinity chromatography purification step. B) A size exclusion chromatography purification step. C) Analytical SDS-PAGE. D) Analytical size exclusion chromatography. The experimental protocol was as described in example 1.

FIG. 19 shows the purification and analysis of wild-type 3F2 human IgG. A) Protein A affinity chromatography purification step. B) A size exclusion chromatography purification step. C) Analytical SDS-PAGE. D) Analytical size exclusion chromatography. The experimental protocol was as described in example 1.

Figure 20 shows purification and analysis of glycoengineered 3F2 human IgG. A) Protein A affinity chromatography purification step. B) A size exclusion chromatography purification step. C) Analytical SDS-PAGE. D) Analytical size exclusion chromatography. The experimental protocol was as described in example 1.

FIG. 21 shows the purification and analysis of wild-type 4G8 human IgG. A) Protein A affinity chromatography purification step. B) A size exclusion chromatography purification step. C) Analytical SDS-PAGE. D) Analytical size exclusion chromatography. The experimental protocol was as described in example 1.

Figure 22 shows purification and analysis of glycoengineered 4G8 human IgG. A) Protein A affinity chromatography purification step. B) A size exclusion chromatography purification step. C) Analytical SDS-PAGE. D) Analytical size exclusion chromatography. The experimental protocol was as described in example 1.

Fig. 23 shows binding of affinity matured anti-FAP antibody 28H1 to human FAP on HEK293 cells compared to the parent 4G8 anti-FAP antibody.

Figure 24 shows the results of an LDH release assay testing for ADCC mediated by wild type (wt) and glycoengineered (ge) forms of the anti-fapp igg antibody 28H1 (affinity matured) and 4G8, 3F8 (parental), HEK 293-hfpp as target cells and PBMNC as effector cells (F/FFc γ RIIIa genotype).

Detailed Description

I. Definition of

For purposes herein, an "acceptor human framework" refers to a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as defined below. An acceptor human framework "derived" from a human immunoglobulin framework or human consensus framework may comprise its identical amino acid sequence, 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 is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

"affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be determined by the dissociation constant (K)_D) Expressed as dissociation and association rate constants (k, respectively)_offAnd k_on) The ratio of. As such, equivalent affinities may comprise different rate constants, as long as the ratio of rate constants remains the same. Affinity can be measured by common methods known in the art, including the methods described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

An "affinity matured" antibody refers to an antibody that has one or more alterations (e.g., amino acid mutations) in one or more hypervariable regions (HVRs) (e.g., CDRs) that result in improved affinity of the antibody for an antigen compared to a parent antibody that does not possess such alterations. Typically, affinity matured antibodies bind to the same epitope as the parent antibody.

The terms "anti-FAP antibody" and "antibody that binds to Fibroblast Activation Protein (FAP)" refer to an antibody that is capable of binding FAP with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent for targeting FAP. In one embodiment, the anti-FAP antibody binds to an unrelated, non-FAP protein to less than about 10% of the binding of the antibody to FAP as measured, for example, by Radioimmunoassay (RIA) or flow cytometry (FACS). In one embodiment, an anti-FAP antibody of the invention binds DPPIV (a protein closely related to FAP, also referred to as CD26, GenBank accession number P27487) to a lesser extent than about 15%, about 10%, or about 5% of the binding of the antibody to FAP, e.g., via FACS measurement. In certain embodiments, an antibody that binds FAP has ≦ 1 μ M ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M) dissociation constant (K)_D). In certain embodiments, the anti-FAP antibody binds to an epitope of FAP that is conserved among FAP from different species.

The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. Also included are antibody fragments having an Fc region, and fusion proteins comprising a region equivalent to an immunoglobulin Fc region.

An "antibody fragment" refers to a molecule distinct from an intact antibody that comprises a portion of the intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab')₂Single chain antibody molecules (e.g., scFv), diabodies, and multispecific antibodies formed from antibody fragments.

An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks binding of the antibody to its antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.

The term "antigen binding domain" refers to the portion of an antigen binding molecule that comprises a region that specifically binds to and is complementary to part or all of an antigen. In the case of larger antigens, the antigen binding molecule may bind only a particular portion of the antigen, which portion is referred to as an epitope. The antigen binding domain may be provided by, for example, one or more antibody variable domains (also referred to as antibody variable regions). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species. For e.g. chimeric antibodies, the non-antigen binding member may be derived from a wide variety of species, including primates such as chimpanzees and humans. Humanized antibodies are a particularly preferred form of chimeric antibody.

The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain possesses. There are 5 major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂The constant domains of heavy chains corresponding to different classes of immunoglobulins are designated α, γ, and μ, respectively.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to: radioisotope (e.g. At)²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²And radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate), doxorubicin (adriamycin), vinca alkaloids (vinca alkaloids) (vincristine), vinblastine (vinblastine), etoposide (etoposide)), doxorubicin (doxorubicin), melphalan (melphalan), mitomycin (mitomycin) C, chlorambucil (chlorembucil), daunorubicin (daunorubicin), or other intercalating agents); a growth inhibitor; enzymes and fragments thereof, such as nucleolytic enzymes; (ii) an antibiotic; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antitumor or anticancer agents disclosed hereinafter.

"Effector function" refers to those biological activities attributable to the Fc region of an antibody and which vary with the antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; secretion of cytokines; immune complex-mediated antigen uptake by antigen presenting cells; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

An "effective amount" of a pharmaceutical agent (e.g., a pharmaceutical formulation) refers to an amount effective to achieve the desired therapeutic or prophylactic result over the necessary dosage and period of time.

The term "Fc region" is used herein to define a 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 (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al, sequence of proteins of immunological interest, 5 th edition public health service, national institutes of health, Bethesda, MD, 1991.

The term "region equivalent to the Fc region of an immunoglobulin" is intended to include naturally occurring allelic variants of the Fc region of an immunoglobulin as well as variants that have alterations that result in substitutions, additions, or deletions, but do not substantially reduce the ability of the immunoglobulin to mediate effector functions, such as antibody-dependent cellular cytotoxicity. For example, one or more amino acids may be deleted from the N-terminus or C-terminus of an immunoglobulin Fc region without substantial loss of biological function. Such variants may be selected according to general rules known in the art so as to have minimal impact on activity. (see, e.g., Bowie, J.U. et al, Science247:1306-10 (1990)).

"framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) (or CDR) residues. In general, the FRs of a variable domain consist of 4 FR domains: FR1, FR2, FR3, and FR 4. Thus, HVR and FR sequences typically occur in the following order in VH (or VL): FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell. In one embodiment, the host cell is engineered to allow for the production of antibodies with modified oligosaccharides. In certain embodiments, the host cell has been further manipulated to express elevated levels of one or more polypeptides having β (1,4) -N-acetylglucosaminyltransferase iii (gntiii) activity. Host cells include cultured cells, for example, mammalian cultured cells such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, per.c6 cells or hybridoma cells, yeast cells, insect cells, plant cells, and the like, and also transgenic animals, transgenic plants, or cells contained within cultured plant or animal tissues.

"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source using a repertoire of human antibodies or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

"human consensus framework" refers to a framework representing the amino acid residues most commonly found in the selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, the sequence subgroups are subgroups as in Kabat et al, sequence of proteins of immunologicalcalemtest, fifth edition, NIHPublication91-3242, BethesdamD (1991), volumes 1-3. In one embodiment, for VL, the subgroup is as in Kabat et al, supra for subgroup kappa I. In one embodiment, for the VH, the subgroup is as in Kabat et al, supra, subgroup III.

A "humanized" antibody is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise at least one, and typically two, substantially the entire variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. Optionally, the humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. An antibody, e.g., a "humanized form" of a non-human antibody, refers to an antibody that has undergone humanization.

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain which is hypervariable in sequence and/or which forms structurally defined loops ("hypervariable loops"). Typically, a native 4 chain antibody comprises 6 HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. In addition to CDR1 in VH, the CDRs generally comprise amino acid residues that form hypervariable loops. "hypervariable regions" (HVRs) are also known as Complementarity Determining Regions (CDRs), and these terms are used interchangeably herein to refer to the variable region portions that form the antigen-binding regions. This particular region has been described by Kabat et al, U.S. department of health and public services ("U.S. Dept. of healthcare and HumanServices)," sequence of proteins of immunological interest "(1983) and by Chothia et al, J.mol.biol.196:901-917(1987), wherein the definitions include overlapping or subsets of amino acid residues when compared to each other. However, any definition refers to the application of the CDRs of an antibody or variant thereof is intended to be within the scope of that term, as defined and used herein. Suitable amino acid residues encompassing the CDRs as defined by each of the references cited above are listed below in table I for comparison. The precise residue number covering a particular CDR will vary with the sequence and size of the CDR. Given the amino acid sequence of the variable region of an antibody, one skilled in the art can routinely determine which residues make up a particular CDR.

Table 1: CDR definition¹

CDR	Kabat	Chothia	AbM2
				VH CDR1	31-35	26-32	26-35
VH CDR2	50-65	52-58	50-58
				VH CDR3	95-102	95-102	95-102
VL CDR1	24-34	26-32	24-34
				VL CDR2	50-56	50-52	50-56
VL CDR3	89-97	91-96	89-97

¹The numbering of all CDR definitions in Table 1 follows the numbering convention set forth by Kabat et al (see below).

²"AbM" with the lower case letter "b" as used in table 1 refers to the CDRs as defined by the "AbM" antibody modeling software of oxford molecular.

Kabat et al also define a numbering system that can be applied to the variable region sequences of any antibody. One of ordinary skill in the art can explicitly assign this system of "Kabat numbering" to any variable region sequence without relying on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth in Kabat et al, United states department of health and public service, "sequence of proteins of immunologicals Interest" (1983). Unless otherwise specified, reference to the numbering of a particular amino acid residue position in the variable region of an antibody is according to the Kabat numbering system.

CDRs also contain "specificity determining residues", or "SDRs", which are residues that contact the antigen. SDR is contained within a CDR region called a shortened-CDR, or a-CDR. Typically, only one fifth to one third of the residues in a given CDR are involved in antigen binding. Specificity determining residues in a particular CDR can be identified by, for example, calculating the interatomic contacts from three-dimensional modeling and determining the sequence variability at a given residue position according to the methods described in Padlan et al, FASEBJ.9(1):133-139 (1995). Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are present at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 50-58 of 31-35B, H2 of H1, and 95-102 of H3 (see Almagro and Fransson, Front. biosci.13:1619-1633 (2008)).

An "antibody conjugate" refers to an antibody conjugated to a cytotoxic agent.

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

An "isolated" antibody refers to an antibody that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessing antibody purity, see, e.g., Flatman et al, J.Chromatogr.B848:79-87 (2007).

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

An "isolated polynucleotide encoding an anti-FAP antibody" refers to one or more polynucleotide molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including such polynucleotide molecules in a single vector or in different vectors, and such polynucleotide molecules present at one or more locations in a host cell.

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 and/or bind the same epitope, except, for example, for possible variant antibodies containing naturally occurring mutations or occurring during the production of a monoclonal antibody preparation, such variants are typically present in very small amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be generated by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for generating monoclonal antibodies are described herein.

"naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radioactive label. The naked antibody may be present in a pharmaceutical formulation.

"Natural antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, a native IgG antibody is an heterotetrameric glycan protein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From N to C-terminus, each heavy chain has one variable region (VH), also known as the variable or heavy chain variable domain, followed by three constant domains (CH 1, CH2, and CH 3), also known as heavy chain constant regions. Similarly, from N-to C-terminus, each light chain has a variable region (VL), also known as the variable light domain or light chain variable domain, followed by a Constant Light (CL) domain, also known as the light chain constant region. Antibody light chains can be classified into one of two types, called kappa (κ) and lambda (λ), based on their constant domain amino acid sequences.

The term "not substantially cross-reactive" means that a molecule (e.g., an antibody) does not recognize or specifically bind to an antigen that is different from the actual target antigen of the molecule (e.g., an antigen that is closely related to the target antigen), particularly when compared to the target antigen. For example, an antibody may bind less than about 10% to less than about 5% of an antigen that is different from the actual target antigen, or may bind an antigen that is different from the actual target antigen in an amount selected from the group consisting of less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%, preferably less than about 2%, 1%, or 0.5% of an antigen that is different from the actual target antigen, and most preferably less than about 0.2% or 0.1% of an antigen that is different from the actual target antigen.

The term "package insert" is used to refer to instructions for use typically contained in commercial packaging for a therapeutic product that contains information regarding the indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings relating to the use of such therapeutic products.

The term "parent" antibody refers to an antibody used as a starting point or basis for making variants.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Comparison for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for purposes of the present invention,% amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, inc, and the source code has been submitted to the us copyright office (USCopyrightOffice, washington d.c.,20559) along with the user document, where it is registered with us copyright registration number TXU 510087. The ALIGN-2 program is publicly available from Genentech, Inc., south san Francisco, Calif., or may be compiled from source code. The ALIGN2 program should be compiled for use on UNIX operating systems, including digital UNIXV4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were not changed.

In the case of employing ALIGN-2 to compare amino acid sequences, the% amino acid sequence identity of a given amino acid sequence a relative to (to), with (with), or against (against) a given amino acid sequence B (or may be stated as having or comprising a given amino acid sequence a with respect to, with, or against a given amino acid sequence B) is calculated as follows:

fractional X/Y times 100

Wherein X is the number of amino acid residues scored as identical matches in the A and B alignments of the sequence alignment program by the program ALIGN-2, and wherein Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence a is not equal to the length of amino acid sequence B, then the% amino acid sequence identity of a relative to B will not equal the% amino acid sequence identity of B relative to a. Unless otherwise specifically indicated, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

Similarly, a nucleic acid or polynucleotide having a nucleotide sequence that is at least, e.g., 95% "identical" to a reference nucleotide sequence of the present invention means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or up to 5% by number of nucleotides of the total nucleotides in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence may occur at the 5 'or 3' end positions of the reference nucleotide sequence or anywhere between those end positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. Indeed, known computer programs (such as those listed above) can be used routinely to determine whether any particular polynucleotide or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence or polypeptide sequence of the present invention.

The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective, and that is free of other components having unacceptable toxicity to a subject that will receive administration of the formulation.

"pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation that is different from the active ingredient and is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, the term "Fibroblast Activation Protein (FAP)" refers to any native FAP from any vertebrate source, including mammals such as primates (e.g., human, see GenBank accession No. AAC51668) and rodents (e.g., mice, see GenBank accession No. AAH19190), unless otherwise indicated. The term encompasses "full-length," unprocessed FAP, and any form of FAP that results from processing in a cell. The term also encompasses naturally occurring variants of FAP, such as splice variants or allelic variants. Preferably, the anti-FAP antibodies of the invention bind to the extracellular domain of FAP. The amino acid sequences of exemplary human, mouse, and cynomolgus FAP extracellular domains (with C-terminal polylysine and 6xHis tags) are shown in seq id no:317, seq id no:319, and seq id no:321, respectively.

As used herein, "treatment" refers to clinical intervention in an attempt to alter the natural course of disease in the individual being treated, and may be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment/diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and regression or improved prognosis. In some embodiments, antibodies of the invention are used to delay the development of or slow the progression of disease.

The term "variable region" or "variable domain" refers to a domain in an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The heavy and light chain variable domains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising 4 conserved Framework Regions (FR) and 3 hypervariable regions (HVRs). (see, e.g., Kindt et al KubyImmunology, 6 th edition, W.H.FreemanndCo., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind a particular antigen can be isolated by screening libraries of complementary VL or VH domains using VH or VL domains, respectively, from antibodies that bind the antigen. See, for example, Portolano et al, J.Immunol.150:880-887(1993); Clarkson et al, Nature352:624-628 (1991).

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures and vectors which integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

As used herein, the term "polypeptide having GnTIII activity" refers to a polypeptide that is capable of catalyzing the addition of N-acetylglucosamine (GlcNAc) residues in β -1-4 linkages to the β -linked mannoside of the trimannosyl core of an N-linked oligosaccharide. This includes fusion polypeptides that exhibit, with or without dose-dependency, an enzymatic activity similar to, but not necessarily identical to, that of beta (1,4) -N-acetylglucosaminyltransferase III (also known as beta-1, 4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase (EC2.4.1.144), according to the international union of biochemistry and molecular biology nomenclature committee (nomenclaturecommittee of nomenclature), NC-IUBMB). Where dose-dependence does exist, it need not be the same as that of GnTIII, but rather is substantially similar to that of a given activity as compared to GnTIII (i.e., the candidate polypeptide will exhibit greater activity or no more than about 25-fold less activity and preferably no more than about 10-fold less activity, and most preferably no more than about 3-fold less activity relative to GnTIII).

As used herein, the term "Golgi localization domain" refers to an amino acid sequence of a Golgi resident polypeptide that is responsible for anchoring the polypeptide to a location within the Golgi complex. Typically, the localization domain comprises the amino-terminal "tail" of the enzyme.

As used herein, the terms "engineered" or "engineered" and the term "glycosylation engineering" with the prefix "sugar" in particular are considered to include any manipulation of the glycosylation pattern of a naturally occurring or recombinant polypeptide or fragment thereof. The glycosylation process involves metabolic engineering of the glycosylation structure of a cell, including genetic manipulation of the oligosaccharide synthesis pathway to achieve altered glycosylation of glycoproteins expressed in the cell. In addition, glycosylation engineering includes mutations and the effects of the cellular environment on glycosylation. In one embodiment, the glycosylation engineering is a change in glycosyltransferase activity. In a specific embodiment, the engineering results in an altered glucosyltransferase activity and/or fucosyltransferase activity.

As used herein, the term "Fc-mediated cellular cytotoxicity" includes antibody-dependent cellular cytotoxicity (ADCC) and cellular cytotoxicity mediated by soluble Fc-fusion proteins containing a human Fc-region. It is an immune mechanism that results in the "lysis" of targeted cells by "human immune effector cells".

As used herein, the term "human immune effector cell" refers to a population of leukocytes which display Fc receptors on their surface, which bind to the Fc region of an antibody or Fc fusion protein through the Fc receptors and perform effector functions. Such populations may include, but are not limited to, Peripheral Blood Mononuclear Cells (PBMCs) and/or Natural Killer (NK) cells.

As used herein, the term "targeted cell" refers to a cell to which an antigen binding molecule (e.g., an antibody or Fc region-containing fragment thereof) or Fc fusion protein that comprises an Fc region specifically binds. The antigen binding molecule or Fc fusion protein binds to the target cell via the protein portion at the N-terminus of the Fc region.

As used herein, the term "increased Fc-mediated cellular cytotoxicity" is defined as an increase in the number of "targeted cells" that are lysed in a given time by a given concentration of antibody or Fc-fusion protein in the medium surrounding the target cells through the mechanism of Fc-mediated cellular cytotoxicity defined above and/or a decrease in the concentration of antibody or Fc-fusion protein in the medium surrounding the target cells required to achieve a given number of "targeted cells" lysis in a given time through the mechanism of Fc-mediated cellular cytotoxicity. The increase in Fc-mediated cellular cytotoxicity is relative to cellular cytotoxicity mediated by the same antigen binding molecule or Fc fusion protein produced by the same type of host cell using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but not by host cells engineered to have an altered glycosylation pattern (e.g., expressing the glycosyltransferase GnTIII, or other glycosyltransferases) by the methods described herein.

By "antibody with increased antibody-dependent cell-mediated cytotoxicity (ADCC)" is meant an antibody (as the term is defined herein) with increased ADCC, as determined by any suitable method known to those of ordinary skill in the art. One recognized in vitro ADCC assay is as follows:

1) the assay uses target cells known to express a target antigen recognized by an antigen binding region of an antibody;

2) the assay uses human Peripheral Blood Mononuclear Cells (PBMCs) isolated from blood of randomly selected healthy donors as effector cells;

3) the assay was performed according to the following protocol:

i) PBMCs were separated using standard density centrifugation protocol and were fractionated at 5x10⁶Each cell/ml in

Suspension in RPMI cell culture medium;

ii) the target cells are cultured by standard tissue culture methods, harvested from exponential growth phase with viability above 90%, washed in RPMI cell culture medium, 100 micro Curie⁵¹Cr labeling, washing twice with cell culture medium and washing at 10 deg.C⁵Resuspend the density of individual cells/ml in cell culture medium;

iii) transferring 100. mu.l of the final target cell suspension to each well of a 96-well microtiter plate;

iv) serially diluting the antibody in cell culture medium from 4000ng/ml to 0.04ng/ml and adding 50 microliters of the resulting antibody solution to the target cells in a 96-well microtiter plate, testing each antibody concentration in triplicate covering the entire concentration range described above;

v) for Maximum Release (MR) control, 3 additional wells in the plate containing labeled target cells received 50 μ l of 2% (V/V) aqueous solution of non-ionic detergent (Nonidet, Sigma, st. louis) instead of antibody solution (point iv above);

vi) for the Spontaneous Release (SR) control, 3 additional wells in the plate containing labeled target cells received 50 microliters of RPMI cell culture medium instead of antibody solution (point iv above);

vii) then, 96-well microtiter plates were centrifuged at 50Xg for 1 minute and incubated at 4 ℃ for 1 hour;

viii) 50. mu.l of PBMC suspension (point i above) was added to each wellTo produce an effector to target ratio of 25:1 and plates were incubated in an incubator at 5% CO₂Standing at 37 ℃ for 4 hours under the atmosphere;

ix) cell-free supernatants from each well were harvested and radioactivity released (ER) from the experiment was quantified using a gamma counter;

x) calculating the percentage of specific lysis for each antibody concentration according to the formula (ER-MR)/(MR-SR) x100, wherein ER is the average radioactivity quantified (see point ix above) for said antibody concentration, MR is the average radioactivity quantified (see point ix above) for the MR control (see point v above) and SR is the average radioactivity quantified (see point ix above) for the SR control (see point vi above);

4) "elevated ADCC" is defined as the increase in the maximum percentage of specific lysis observed over the range of antibody concentrations tested above and/or the decrease in antibody concentration required to reach half the maximum percentage of specific lysis observed over the range of antibody concentrations tested above. The increase in ADCC is relative to ADCC mediated by the same antibody produced by the same type of host cell, but not by the same host cell engineered to overexpress GnTIII, as measured by the above assay, using the same standard production, purification, formulation and storage methods known to those skilled in the art.

Compositions and methods

Fibroblast Activation Protein (FAP) is expressed in most tumors but is essentially absent in healthy adult tissues, and thus antibodies targeting this antigen have great therapeutic potential. The present invention provides antibodies that bind FAP, particularly antibodies with high affinity and strong effector function. The antibodies of the invention are useful, for example, in the diagnosis and treatment of diseases characterized by FAP expression, such as cancer.

A. Exemplary anti-FAP antibodies

The present invention provides antibodies that specifically bind to Fibroblast Activation Protein (FAP). In particular, the invention provides antibodies that specifically bind FAP, wherein the antibodies are glycoengineered to have increased effector function.

In one embodiment, an anti-FAP antibody of the invention comprises at least one (e.g., one, two, three, four, five, or six) heavy or light chain Complementarity Determining Region (CDR) selected from the group consisting of: SEQ ID NO. 3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61, SEQ ID NO. 63, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO. 73, SEQ ID NO. 99, SEQ ID NO. 95, SEQ ID NO. 99, SEQ ID NO. 95, SEQ ID NO. 99, SEQ ID NO. 33, SEQ ID NO. 95, SEQ ID NO, 133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175, 177 or variants or truncated forms of the aforementioned sequences (which contain at least a Specificity Determining Residue (SDR)) as the CDR.

In one embodiment, the at least one CDR is a heavy chain CDR, particularly heavy chain CDR3 selected from SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, and SEQ ID NO: 141. In another embodiment, the antibody comprises at least one heavy chain CDR and at least one light chain CDR, particularly heavy chain CDR3 selected from SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, and SEQ ID NO:141 and light chain CDR3 selected from SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, and SEQ ID NO: 177.

In one embodiment, the antibody of the invention comprises at least one, at least two, or all three heavy chain cdr (HCDR) sequences selected from the group consisting of (a) HCDR1 comprising an amino acid sequence selected from the group consisting of seq id No. 3, seq id No.5, seq id No.7, seq id No. 9, seq id No. 11, seq id No.13, seq id No. 15, seq id No. 17, seq id No. 19, seq id No. 21, seq id No. 23, seq id No. 25, seq id No. 27, seq id No. 29, seq id No. 31, and seq id No. 33; (b) 35,37,39, 51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99, 119, 95, 119, 97, 119, 103, 89, 105,107, 119, 97, 119, 107, 119,121, 131, 123; and (c) HCDR3 comprising an amino acid sequence selected from SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, and SEQ ID NO: 141. In yet another embodiment, the antibody comprises a heavy chain variable region comprising (a) heavy chain CDR1 selected from the group consisting of seq id No. 3, seq id No.5, seq id No.7, seq id No. 9, seq id No. 11, seq id No.13, seq id No. 15, seq id No. 17, seq id No. 19, seq id No. 21, seq id No. 23, seq id No. 25, seq id No. 27, seq id No. 29, seq id No. 31, and seq id No. 33; (b) 35,37,39, 51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99, 127, 119, 97, 119, 103,105,107, 119, 97, 119, 107, 119, 121; and (c) a heavy chain CDR3 selected from SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, and SEQ ID NO:141, or a variant or truncated form of the above sequence (which contains at least SDR) as said CDR.

In one embodiment, an antibody of the invention comprises at least one, at least two, or all three light chain cdr (LCDR) sequences selected from the group consisting of (a) LCDR1 comprising an amino acid sequence selected from the group consisting of seq id No. 143, seq id No. 145, seq id No. 147, and seq id No. 149; (b) LCDR2 comprising an amino acid sequence selected from SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, and SEQ ID NO: 161; and (c) an LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, and SEQ ID NO: 177. In yet another embodiment, the antibody comprises a light chain variable region comprising (a) light chain CDR1 selected from the group consisting of SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, and SEQ ID NO: 149; (b) a light chain CDR2 selected from SEQ ID NO. 151, SEQ ID NO. 153, SEQ ID NO. 155, SEQ ID NO. 157, SEQ ID NO. 159, and SEQ ID NO. 161; and (c) a light chain CDR3 selected from the group consisting of SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, and SEQ ID NO:177, or a variant or truncated form of the above sequence (which contains at least SDR) as said CDR.

In a more specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising heavy chain CDR1 selected from the group consisting of seq id No. 3, seq id No.5, seq id No.7, seq id No. 9, seq id No. 11, seq id No.13, seq id No. 15, seq id No. 17, seq id No. 19, seq id No. 21, seq id No. 23, seq id No. 25, seq id No. 27, seq id No. 29, seq id No. 31, and seq id No. 33; 35,37,39, 51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99, 127, 119, 97, 119, 103,105,107, 119, 97, 119, 107, 119, 121; and a heavy chain CDR3 selected from SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, and SEQ ID NO:141, the light chain variable region comprising a light chain CDR1 selected from SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, and SEQ ID NO: 149; a light chain CDR2 selected from SEQ ID NO. 151, SEQ ID NO. 153, SEQ ID NO. 155, SEQ ID NO. 157, SEQ ID NO. 159, and SEQ ID NO. 161; and a light chain CDR3 selected from the group consisting of SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, and SEQ ID NO:177, or a variant or truncated form of the above sequence (which contains at least SDR) as the CDR.

In another embodiment, the antibody of the invention comprises a heavy chain variable region comprising heavy chain CDR1 selected from the group consisting of seq id No. 3, seq id No.5, seq id No.7, seq id No. 9, seq id No. 11, seq id No.13, seq id No. 15, seq id No. 17, seq id No. 19, seq id No. 21, seq id No. 23, seq id No. 25, seq id No. 27, seq id No. 29, seq id No. 31, and seq id No. 33; 35,37,39, 51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99, 127, 119, 97, 119, 103,105,107, 119, 97, 119, 107, 119, 121; and a heavy chain CDR3 selected from SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, and SEQ ID NO:141, the light chain variable region comprising a light chain CDR1 selected from SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, and SEQ ID NO: 149; a light chain CDR2 selected from SEQ ID NO. 151, SEQ ID NO. 153, SEQ ID NO. 155, SEQ ID NO. 157, SEQ ID NO. 159, and SEQ ID NO. 161; and a light chain CDR3 selected from the group consisting of SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, and SEQ ID NO:177, wherein at least one of said CDRs is selected from the group consisting of: SEQ ID NO.7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61, SEQ ID NO. 63, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 77, SEQ ID NO. 79, SEQ ID NO. 81, SEQ ID NO. 83, SEQ ID NO. 85, SEQ ID NO. 87, SEQ ID NO. 89, SEQ ID NO. 91, SEQ ID NO. 93, SEQ ID NO. 95, SEQ ID NO. 97, SEQ ID NO. 99, SEQ ID NO. 109, SEQ ID NO. 111, SEQ ID NO. 115, SEQ ID NO. 127, SEQ ID NO. 123, SEQ ID NO. 129, SEQ ID NO. 131, SEQ ID NO. 123, SEQ ID NO. 129, and SEQ ID NO. 177.

In another embodiment, the antibody of the invention comprises a heavy chain variable region comprising heavy chain CDR1 selected from the group consisting of seq id No. 3, seq id No.5, seq id No.7, seq id No. 9, seq id No. 11, seq id No.13, seq id No. 15, seq id No. 17, seq id No. 19, seq id No. 21, seq id No. 23, seq id No. 25, seq id No. 27, seq id No. 29, seq id No. 31, and seq id No. 33; 35,37,39, 51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99, 127, 119, 97, 119, 103,105,107, 119, 97, 119, 107, 119, 121; and a heavy chain CDR3 selected from SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, and SEQ ID NO:141, the light chain variable region comprising a light chain CDR1 selected from SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, and SEQ ID NO: 149; a light chain CDR2 selected from SEQ ID NO. 151, SEQ ID NO. 153, SEQ ID NO. 155, SEQ ID NO. 157, SEQ ID NO. 159, and SEQ ID NO. 161; and a light chain CDR3 selected from the group consisting of SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, and SEQ ID NO:177, wherein at least one of said CDRs is not a CDR selected from the group consisting of: SEQ ID NO. 3, SEQ ID NO.5, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO. 73, SEQ ID NO. 75, SEQ ID NO. 101, SEQ ID NO. 103, SEQ ID NO. 105, SEQ ID NO. 107, SEQ ID NO. 135, SEQ ID NO. 137, SEQ ID NO. 139, SEQ ID NO. 141, SEQ ID NO. 143, SEQ ID NO. 145, SEQ ID NO. 147, SEQ ID NO. 149, SEQ ID NO. 151, SEQ ID NO. 153, SEQ ID NO. 155, SEQ ID NO. 157, SEQ ID NO. 159, SEQ ID NO. 161, SEQ ID NO. 163, SEQ ID NO. 167, SEQ ID NO. 165, SEQ ID NO. 175, SEQ ID NO. 171, SEQ ID NO. 175, SEQ ID NO. 169, and SEQ ID NO. 171.

In another embodiment, the antibody of the invention comprises a heavy chain variable region comprising heavy chain CDR1 selected from the group consisting of seq id No. 3, seq id No.5, seq id No.13, seq id No. 15, seq id No. 23, seq id No. 25, and seq id No. 27; heavy chain CDR2 selected from SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 69, SEQ ID NO. 71, SEQ ID NO. 73, SEQ ID NO. 75, SEQ ID NO. 101, SEQ ID NO. 103, SEQ ID NO. 105, and SEQ ID NO. 107; and a heavy chain CDR3 selected from SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, and SEQ ID NO:141, the light chain variable region comprising a light chain CDR1 selected from SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, and SEQ ID NO: 149; a light chain CDR2 selected from SEQ ID NO. 151, SEQ ID NO. 153, SEQ ID NO. 155, SEQ ID NO. 157, SEQ ID NO. 159, and SEQ ID NO. 161; and a light chain CDR3 selected from the group consisting of SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, and SEQ ID NO:175, or a variant or truncated form of the above sequence (which contains at least SDR) as the CDR.

In a specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising heavy chain CDR1 selected from seq id No. 3, seq id No.13, and seq id No. 23; a heavy chain CDR2 selected from SEQ ID NO. 35, SEQ ID NO. 69, and SEQ ID NO. 101; and heavy chain CDR3SEQ ID NO:135, the light chain variable region comprising light chain CDR1SEQ ID NO:143, light chain CDR2SEQ ID NO: 151; and light chain CDR3SEQ ID NO: 163. In another specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising heavy chain CDR1 selected from the group consisting of seq id No. 3, seq id No.13, and seq id No. 23; a heavy chain CDR2 selected from SEQ ID NO:37, SEQ ID NO:71, and SEQ ID NO: 103; and heavy chain CDR3SEQ ID NO:137, the light chain variable region comprising light chain CDR1SEQ ID NO:145, light chain CDR2SEQ ID NO: 153; and light chain CDR3SEQ ID NO: 165. In yet another specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising heavy chain CDR1 selected from the group consisting of seq id No. 3, seq id No.13, and seq id No. 23; a heavy chain CDR2 selected from SEQ ID NO. 35, SEQ ID NO. 69, and SEQ ID NO. 101; and heavy chain CDR3SEQ ID NO:137, the light chain variable region comprising light chain CDR1SEQ ID NO:147, light chain CDR2SEQ ID NO: 155; and light chain CDR3SEQ ID NO: 167. In another specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising heavy chain CDR1 selected from the group consisting of seq id No. 3, seq id No.13, and seq id No. 23; a heavy chain CDR2 selected from SEQ ID NO:39, SEQ ID NO:73, and SEQ ID NO: 105; and heavy chain CDR3SEQ ID NO:135, the light chain variable region comprising light chain CDR1SEQ ID NO:145, light chain CDR2SEQ ID NO: 153; and light chain CDR3SEQ ID NO: 169. In another specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising heavy chain CDR1 selected from the group consisting of seq id No. 3, seq id No.13, and seq id No. 23; a heavy chain CDR2 selected from SEQ ID NO. 35, SEQ ID NO. 69, and SEQ ID NO. 101; and heavy chain CDR3SEQ ID NO:137, the light chain variable region comprising light chain CDR1SEQ ID NO:149, light chain CDR2SEQ ID NO: 157; and light chain CDR3SEQ ID NO: 167. In another specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising heavy chain CDR1 selected from the group consisting of seq id No. 3, seq id No.7, seq id No.13, seq id No. 17, seq id No. 23, and seq id No. 29; (iii) a heavy chain CDR2 selected from SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49, SEQ ID NO 51, SEQ ID NO 53, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 63, SEQ ID NO 65, SEQ ID NO 77, SEQ ID NO 79, SEQ ID NO 81, SEQ ID NO 83, SEQ ID NO 85, SEQ ID NO 87, SEQ ID NO 89, SEQ ID NO91, SEQ ID NO 93, SEQ ID NO 97, SEQ ID NO 109, SEQ ID NO 111, SEQ ID NO 113, SEQ ID NO 115, SEQ ID NO 117, SEQ ID NO 119, SEQ ID NO 121, SEQ ID NO 123, SEQ ID NO 125, SEQ ID NO 129, and SEQ ID NO 131; and heavy chain CDR3SEQ ID NO:135, the light chain variable region comprising light chain CDR1SEQ ID NO:143, light chain CDR2SEQ ID NO: 151; and light chain CDR3SEQ ID NO: 163. In yet another specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising heavy chain CDR1 selected from the group consisting of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:31, and SEQ ID NO: 33; heavy chain CDR2 selected from SEQ ID NO. 61, SEQ ID NO. 67, SEQ ID NO. 95, SEQ ID NO. 99, SEQ ID NO. 127, and SEQ ID NO. 133; and heavy chain CDR3SEQ ID NO:137, the light chain variable region comprising light chain CDR1SEQ ID NO:147, light chain CDR2SEQ ID NO: 155; and light chain CDR3SEQ ID NO: 167. In another specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising heavy chain CDR1 selected from the group consisting of seq id No. 3, seq id No.13, and seq id No. 23; a heavy chain CDR2 selected from SEQ ID NO. 35, SEQ ID NO. 69, and SEQ ID NO. 101; and heavy chain CDR3SEQ ID NO:135, the light chain variable region comprising light chain CDR1SEQ ID NO:143, light chain CDR2SEQ ID NO: 151; and light chain CDR3SEQ ID NO: 177. In yet another specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising heavy chain CDR1 selected from the group consisting of seq id No. 3, seq id No.13, and seq id No. 23; a heavy chain CDR2 selected from SEQ ID NO:43, SEQ ID NO:77, and SEQ ID NO: 109; and heavy chain CDR3SEQ ID NO:135, the light chain variable region comprising light chain CDR1SEQ ID NO:143, light chain CDR2SEQ ID NO: 151; and light chain CDR3SEQ ID NO: 163. In yet another specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising heavy chain CDR1 selected from the group consisting of seq id No. 3, seq id No.13, and seq id No. 23; a heavy chain CDR2 selected from SEQ ID NO:45, SEQ ID NO:79, and SEQ ID NO: 111; and heavy chain CDR3SEQ ID NO:135, the light chain variable region comprising light chain CDR1SEQ ID NO:143, light chain CDR2SEQ ID NO: 151; and light chain CDR3SEQ ID NO: 163. In yet another specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising heavy chain CDR1 selected from the group consisting of seq id No. 3, seq id No.13, and seq id No. 23; a heavy chain CDR2 selected from SEQ ID NO. 65, SEQ ID NO. 89, and SEQ ID NO. 131; and heavy chain CDR3SEQ ID NO:135, the light chain variable region comprising light chain CDR1SEQ ID NO:143, light chain CDR2SEQ ID NO: 151; and light chain CDR3SEQ ID NO: 163. In yet another specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising heavy chain CDR1 selected from the group consisting of seq id No. 3, seq id No.13, and seq id No. 23; a heavy chain CDR2 selected from SEQ ID NO. 47, SEQ ID NO. 81, and SEQ ID NO. 113; and heavy chain CDR3SEQ ID NO:135, the light chain variable region comprising light chain CDR1SEQ ID NO:143, light chain CDR2SEQ ID NO: 151; and light chain CDR3SEQ ID NO: 163. In yet another specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising heavy chain CDR1 selected from the group consisting of seq id No. 9, seq id No. 19, and seq id No. 31; a heavy chain CDR2 selected from SEQ ID NO:61, SEQ ID NO:95, and SEQ ID NO: 127; and heavy chain CDR3SEQ ID NO:137, the light chain variable region comprising light chain CDR1SEQ ID NO:147, light chain CDR2SEQ ID NO: 155; and light chain CDR3SEQ ID NO: 167.

In one embodiment, an antibody of the invention comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least about 90%, 91%, 92%, 94%, 96%, 97%, or 97% identity to a sequence selected from SEQ ID NO:197, 201,203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 299, 303, 307,311, and 97%. In one embodiment, the antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:197, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:207, SEQ ID NO:211, SEQ ID NO:215, SEQ ID NO:219, SEQ ID NO:223, SEQ ID NO:227, SEQ ID NO:231, SEQ ID NO:235, SEQ ID NO:239, SEQ ID NO:243, SEQ ID NO:247, SEQ ID NO:251, SEQ ID NO:255, SEQ ID NO:259, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:271, SEQ ID NO:275, SEQ ID NO:279, SEQ ID NO:283, SEQ ID NO:287, SEQ ID NO:291, SEQ ID NO:295, SEQ ID NO:299, SEQ ID NO:303, SEQ ID NO:307, and SEQ ID NO: 311.

In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-FAP antibody comprising that sequence retains the ability to bind FAP. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in seq id nos 197, 201,203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307, or 311. In certain embodiments, substitutions, insertions, or deletions occur in regions outside of the HVRs or CDRs (i.e., in the FRs). Optionally, the anti-FAP antibody according to the invention comprises the VH sequence in seq id no197, 201,203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307 or 311, including post-translational modifications of this sequence. In a particular embodiment, the VH comprises 1,2 or 3 heavy chain CDRs as HCDR1, HCDR2 and HCDR3 selected from the group consisting of seq id nos3, 5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139 and 141.

In another embodiment, the antibody of the invention comprises a light chain variable region comprising amino acids having at least about 90%, 91%, 92%, 93%, 94%, 96%, 97%, or 99% identity to a sequence selected from seq id No. 193, seq id No. 195, seq id No. 199, seq id No. 205, seq id No. 209, seq id No. 213, seq id No. 217, seq id No. 221, seq id No. 225, seq id No. 229, seq id No. 233, seq id No. 237, seq id No. 241, seq id No. 245, seq id No. 249, seq id No. 253, seq id No. 257, seq id No. 261, seq id No. 265, seq id No. 269, seq id No. 273, seq id No. 277, seq id No. 281, seq id No. 285, seq id No. 289, seq id No. 293, seq id No. 297, seq id No. 301, seq id No. 305, and seq id No. 309. In yet another embodiment, the antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of seq id No. 193, seq id No. 195, seq id No. 199, seq id No. 205, seq id No. 209, seq id No. 213, seq id No. 217, seq id No. 221, seq id No. 225, seq id No. 229, seq id No. 233, seq id No. 237, seq id No. 241, seq id No. 245, seq id No. 249, seq id No. 253, seq id No. 257, seq id No. 261, seq id No. 265, seq id No. 269, seq id No. 273, seq id No. 277, seq id No. 281, seq id No. 285, seq id No. 289, seq id No. 293, seq id No. 297, seq id No. 301, seq id No. 305, and seq id No. 309.

In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-FAP antibody comprising that sequence retains the ability to bind FAP. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in seq id nos 193,195, 199, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265,269, 273, 277, 281, 285, 289, 293, 297, 301, 305, or 309. In certain embodiments, substitutions, insertions, or deletions occur in regions outside of the HVRs or CDRs (i.e., in the FRs). Optionally, an anti-FAP antibody of the invention comprises the VL sequence of seq id no193,195, 199, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265,269, 273, 277, 281, 285, 289, 293, 297, 301, 305, or 309, including post-translational modifications of the sequence. In a particular embodiment, the VL comprises 1,2 or 3 light chain CDRs selected from the group consisting of sequences shown as seq id nos143, 145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175 and 177 as LCDR1, LCDR2 and LCDR 3.

In another aspect, an anti-FAP antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises a heavy chain variable region comprising an amino acid sequence that is identical to at least about 90%, 91%, 92%, 93%, 94%, 95%, 97%, 209%, 223%, 227%, 231%, 235%, 239%, 243%, 247%, 251, 255%, 259%, 263, 267%, 275%, 279, 283, 287, 291, 295, 299, 303%, 195%, 303%, 95%, 225%, 221%, 213%, 303%, 307, 311%, 213%, 303%, 307, and 311%, or a light chain variable region comprising an amino acid sequence that is identical to at least about 90%, 91%, 93%, 94%, 95%, 97%, 225%, 199%, 213%, 199%, or 213%, or a light chain variable region selected from SEQ ID, 237, 241, 245, 249, 253, 257, 261, 265,269, 273, 277, 281, 285, 289, 293, 297, 301, 305, and 309, respectively, of amino acid sequence, which is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In one embodiment, the antibody comprises VH and VL sequences in seq id nos 197, 201,203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307, or 311 and seq id nos 193,195, 199, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265,269, 273, 277, 281, 285, 293, 297, 301, 305, or 309, respectively, including post-translational modifications of those sequences.

In one embodiment, the antibody comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:197, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:207, SEQ ID NO:211, SEQ ID NO:215, SEQ ID NO:219, SEQ ID NO:223, SEQ ID NO:227, SEQ ID NO:231, SEQ ID NO:235, SEQ ID NO:239, SEQ ID NO:243, SEQ ID NO:247, SEQ ID NO:251, SEQ ID NO:255, SEQ ID NO:259, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:271, SEQ ID NO:275, SEQ ID NO:279, SEQ ID NO:283, SEQ ID NO:287, SEQ ID NO:291, SEQ ID NO:295, SEQ ID NO:299, SEQ ID NO:303, SEQ ID NO:307, SEQ ID NO:311, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:225, SEQ ID NO: 185, SEQ ID NO:193, SEQ ID NO: 185, SEQ ID NO:225, SEQ ID NO:85, SEQ ID NO: 185, SEQ ID NO:85, SEQ ID NO:11, SEQ, 277, 281, 285, 289, 293, 297, 301, 305, and 309, wherein at least one of the variable regions does not comprise an amino acid sequence selected from the group consisting of seq id No. 193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235, 239, 253, 251.

In one embodiment, the antibody of the invention comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:197, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:207, SEQ ID NO:211, SEQ ID NO:215, SEQ ID NO:219, SEQ ID NO:223, SEQ ID NO:227, SEQ ID NO:231, SEQ ID NO:235, SEQ ID NO:239, SEQ ID NO:243, SEQ ID NO:247, SEQ ID NO:251, SEQ ID NO:255, SEQ ID NO:259, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:241, SEQ ID NO:275, SEQ ID NO:279, SEQ ID NO:283, SEQ ID NO:287, SEQ ID NO:291, SEQ ID NO:295, SEQ ID NO:299, SEQ ID NO:303, SEQ ID NO:307, and SEQ ID NO:311, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:225, SEQ ID NO: 185, SEQ ID NO:85, SEQ ID NO:241, SEQ ID NO:85, SEQ ID NO: 185, SEQ ID, 277, 281, 285, 289, 293, 297, 301, 305, and 309, and at least one of the variable regions comprises an amino acid sequence selected from the group consisting of seq id No. 259, 263, 267, 271, 275, 279, 283, 287, 291,293, 299, 303, 307, and 311.

In one embodiment, the antibody comprises a heavy chain variable region comprising a heavy chain variable region that differs from a light chain variable region selected from seq id nos: 197. SEQ ID NO: 201. SEQ ID NO: 203. SEQ ID NO: 207. SEQ ID NO: 211. SEQ ID NO: 215. SEQ ID NO: 219. SEQ ID NO: 223. SEQ ID NO: 227. SEQ ID NO: 231. SEQ ID NO: 235. SEQ ID NO: 239. SEQ ID NO: 243. SEQ ID NO: 247. SEQ ID NO: 251. and SEQ ID NO:255, an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, the light chain variable region comprises a light chain variable region and a light chain variable region selected from the group consisting of SEQ ID NOs: 193. SEQ ID NO: 195. SEQ ID NO: 199. SEQ ID NO: 205. SEQ ID NO: 209. SEQ ID NO: 213. SEQ ID NO: 217. SEQ ID NO: 221. SEQ ID NO: 225. SEQ ID NO: 229. SEQ ID NO: 233. SEQ ID NO: 237. SEQ ID NO: 241. SEQ ID NO: 245. SEQ ID NO: 249. and SEQ ID NO:253, or a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

In a specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:197 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:193 or SEQ ID NO: 195. In another specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:201 or SEQ ID NO:203 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 199. In yet another specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:207 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 205. In another specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:211 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 209. In yet another specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:219 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 217. In another embodiment, the antibody of the invention comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:259, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:271, SEQ ID NO:275, SEQ ID NO:279, SEQ ID NO:283, SEQ ID NO:287, SEQ ID NO:291, SEQ ID NO:299, SEQ ID NO:303, SEQ ID NO:307, and SEQ ID NO:311, or a light chain variable region comprising the amino acid sequence SEQ ID NO: 293. In a specific embodiment, the antibody of the invention comprises a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of 259, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:271, SEQ ID NO:275, SEQ ID NO:279, SEQ ID NO:283, SEQ ID NO:287, SEQ ID NO:291, SEQ ID NO:303, and SEQ ID NO:307, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 195; or b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:299 or SEQ ID NO:311 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 205; or c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:197 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 293. In a specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising the amino acid sequence of seq id No. 259 and a light chain variable region comprising the amino acid sequence of seq id No. 195. In another specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:263 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 195. In a specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising the amino acid sequence of seq id No. 307 and a light chain variable region comprising the amino acid sequence of seq id No. 305. In a specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:267 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 265. In another specific embodiment, the antibody of the invention comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:299 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 205. In a particular embodiment, the antibody according to any of the above embodiments further comprises an Fc region or a region equivalent to an immunoglobulin Fc region.

In one embodiment, the antibody of the invention comprises an Fc region, particularly an IgGFc region, most particularly an IgG1Fc region.

In a particular embodiment, the antibody of the invention is a full length antibody, particularly an IgG class antibody, most particularly an IgG1 isotype antibody. In another embodiment, the antibody of the invention is an antibody fragment selected from the group consisting of: scFv fragments, Fv fragments, Fab fragments, and F (ab') 2 fragments. In yet another embodiment, the antibody of the invention is an antibody fragment having an Fc region, or a fusion protein comprising a region equivalent to an Fc region of an immunoglobulin. In one embodiment, the antibody of the invention is a monoclonal antibody.

In one embodiment, the antibody of the invention is chimeric, most particularly humanized. In a particular embodiment, the antibodies of the invention are human. In another embodiment, an antibody of the invention comprises a human constant region. In one embodiment, the antibody of the invention comprises a human Fc region, particularly a human IgGFc region, most particularly a human IgG1Fc region.

In one embodiment, the antibody of the invention comprises a heavy chain constant region, wherein said heavy chain constant region is a human IgG constant region, particularly a human IgG1 constant region, comprising an Fc region. In a specific embodiment, the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 313. In another specific embodiment, an antibody of the invention comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 315. In yet another specific embodiment, an antibody of the invention comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO. 313 and a light chain constant region comprising the amino acid sequence of SEQ ID NO. 315.

In a particular embodiment, the invention provides an antibody that specifically binds FAP, wherein the antibody comprises a) a heavy chain variable region or a light chain variable region comprising a variable region identical to at least one amino acid sequence selected from seq id No. 197, seq id No. 201, seq id No. 203, seq id No. 207, seq id No. 211, seq id No. 215, seq id No. 219, seq id No. 223, seq id No. 227, seq id No. 231, seq id No. 235, seq id No. 239, seq id No. 243, seq id No. 247, seq id No. 251, seq id No. 255, seq id No. 259, seq id No. 263, seq id No. 267, seq id No. 271, seq id No. 275, seq id No. 279, seq id No. 287, seq id No. 291, seq id No. 295, seq id No. 299, seq id No. 303, seq id No. 307, seq id No. 283, seq id No. 193, seq id No. 92, seq id No. 91, seq id No. 95%, seq id No. 97%, seq id No. 209, seq id No. 205, seq id No. 100%, or light chain variable region comprising at least one or more than one amino acid sequence selected from seq id No. 95, seq id No. 100, seq id No.1, seq, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265,269, 273, 277, 281, 285, 289, 293, 297, 293, 297, 301, 305, 309, 96, 97, 98, 99 or 100% identical amino acid sequences or any combination of the above heavy and light chain variable regions and b) an Fc region or a region equivalent to an immunoglobulin Fc region.

In one embodiment, the antibody of the invention comprises an Fc region, wherein the Fc region is a glycoengineered Fc region. In yet another embodiment, the antibody of the invention is glycoengineered to have a modified oligosaccharide in the Fc region. In a specific embodiment, the antibody has an increased proportion of bisected oligosaccharides in the Fc region as compared to the non-glycoengineered antibody. In a more specific embodiment, at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, preferably at least about 50%, more preferably at least about 70% of the N-linked oligosaccharides in the Fc region of the antibody are bisected. The bisected oligosaccharides may be of the hybrid or complex type.

In another specific embodiment, the antibodies of the invention have an increased proportion of nonfucosylated oligosaccharides in the Fc region as compared to the nonglycoengineered antibody. In a more specific embodiment, at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, preferably at least about 50%, more preferably at least about 70% of the N-linked oligosaccharides in the Fc region of the antibody are nonfucosylated. The nonfucosylated oligosaccharides may be of the hybrid or complex type.

In a particular embodiment, the antibodies of the invention have an increased proportion of bisected, nonfucosylated oligosaccharides in the Fc region as compared to the nonglycoengineered antibodies. Specifically, the antibody comprises an Fc region wherein at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, preferably at least about 15%, more preferably at least about 25%, at least about 35%, or at least about 50% of the N-linked oligosaccharides are bisected, nonfucosylated. The bisected, nonfucosylated oligosaccharides may be of the hybrid or complex type.

In one embodiment, the antibodies of the invention have increased effector function and/or increased Fc receptor binding affinity. Increased effector function and/or increased Fc receptor binding may result, for example, from glycoengineering and/or affinity maturation of the antibody. In one embodiment, increased effector function and/or increased Fc receptor binding is the result of glycoengineering of the Fc region of the antibody. In another embodiment, increased effector function and/or increased Fc receptor binding is the result of a combination of increased affinity and glycoengineering. The increased effector function may include, but is not limited to, one or more of the following: increased Fc-mediated cellular cytotoxicity (including increased antibody-dependent cell-mediated cytotoxicity (ADCC)), increased antibody-dependent cellular phagocytosis (ADCP), increased cytokine secretion, increased immune complex-mediated antigen uptake by antigen presenting cells, increased binding to NK cells, increased binding to macrophages, increased binding to monocytes, increased binding to polymorphonuclear cells, increased direct apoptosis-inducing signaling, increased target-bound antibody cross-linking, increased dendritic cell maturation, or increased T cell priming. In a particular embodiment, the increased effector function is increased ADCC. Increased Fc receptor binding preferably increases binding to Fc activating receptors, most preferably Fc γ RIIIa.

In one embodiment, the antibodies of the invention do not cause clinically significant levels of toxicity when administered to an individual in a therapeutically effective amount.

In one embodiment, the antibodies of the invention are affinity matured. In yet another embodiment, an antibody of the invention has a dissociation constant (K) of less than about 1 μ M to about 0.001nM_D) Value ofIn particular, a K of less than about 100nM, less than about 10nM, less than about 1nM, or less than about 0.1nM_DThe value is combined to form the fibroblast activation protein. In one embodiment, an antibody of the invention binds to human, mouse, or cynomolgus FAP. In one embodiment, the antibodies of the invention bind to human and cynomolgus FAP. In a more specific embodiment, the antibodies of the invention have a K of less than about 200nM, less than about 100nM, more particularly less than about 10nM or less than about 1nM, most particularly less than about 0.1nM_DValues bind to human, mouse and cynomolgus FAP. Determination of K by surface plasmon resonance using antibodies in the form of Fab or IgG, in particular IgG_DThe value is obtained.

In one embodiment, an anti-FAP antibody of the invention binds FAP in human tissue.

In one embodiment, an anti-FAP antibody of the invention is cross-reactive to human and murine FAP. In another embodiment, the antibodies of the invention are not substantially cross-reactive with other members of the dipeptidyl peptidase IV family, particularly with DPPIV/CD 26. In one embodiment, an anti-FAP antibody of the invention does not induce FAP internalization when said antibody binds FAP expressed on the surface of a cell.

In a particular embodiment, the invention provides an antibody that specifically binds FAP, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence selected from seq id No. 197, seq id No. 201, seq id No. 203, seq id No. 207, seq id No. 211, seq id No. 215, seq id No. 219, seq id No. 223, seq id No. 227, seq id No. 231, seq id No. 235, seq id No. 239, seq id No. 243, seq id No. 247, seq id No. 251, seq id No. 255, seq id No. 259, seq id No. 263, seq id No. 267, seq id No. 271, seq id No. 275, seq id No. 279, seq id No. 283, seq id No. 287, seq id No. 291, seq id No. 295, seq id No. 303, seq id No. 307, seq id No. 193, seq id No. 221, seq id No. 195, seq id No. 213, seq id No. 11, seq id no, 253, 257, 261, 265,269, 273, 277, 281, 285, 289, 293, 297, 301, 305 and 309, and wherein optionally the antibody is glycoengineered to have increased effector function and/or Fc receptor binding affinity. In another particular embodiment, the invention provides an antibody that specifically binds FAP, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence selected from seq id No. 197, seq id No. 201, seq id No. 203, seq id No. 207, seq id No. 211, seq id No. 215, seq id No. 219, seq id No. 223, seq id No. 227, seq id No. 231, seq id No. 235, seq id No. 239, seq id No. 243, seq id No. 247, seq id No. 251, seq id No. 255, seq id No. 259, seq id No. 263, seq id No. 267, seq id No. 271, seq id No. 275, seq id No. 279, seq id No. 283, seq id No. 287, seq id No. 291, seq id No. 295, seq id No. 303, seq id No. 307, seq id No. 193, seq id No. 221, seq id No. 213, seq id no, 249, 253, 257, 261, 265,269, 273, 277, 281, 285, 289, 293, 297, 301, 305 and 309, and wherein the antibody has an amino acid sequence of non-fucosylated oligosaccharides with an increased proportion in the Fc region and/or bisected oligosaccharides with an increased proportion in the Fc region.

In one aspect, the invention provides an antibody that specifically binds FAP, wherein the antibody is derived from a parent antibody comprising heavy chain CDR1seq id No. 3, heavy chain CDR2seq id No. 35, heavy chain CDR3 selected from seq id No. 135, seq id No. 137, seq id No. 139 and seq id No. 141, light chain CDR1seq id No. 145, light chain CDR2seq id No. 153, and light chain CDR3 selected from seq id No. 165, seq id No. 167, seq id No. 169, seq id No. 171, seq id No. 173 and seq id No. 175, and wherein the antibody comprises at least one amino acid substitution or deletion in at least one of the heavy or light chain CDRs of the parent antibody. For example, the antibody may comprise at least one, such as about one to about ten (i.e., about 1,2, 3,4, 5,6, 7,8, 9, or 10), and particularly about two to about five substitutions in one or more hypervariable regions or CDRs (i.e., 1,2, 3,4, 5, or6 hypervariable regions or CDRs) of the parent antibody. In certain embodiments, any one or more amino acids of the parent antibody provided above are substituted or deleted at the following CDR positions:

heavy chain CDR1(SEQ ID NO: 3): 2 and 3 positions

Heavy chain CDR2(SEQ ID NO: 35): 1,3, 4,5, 6,7, 8 and 9 th bit

Light chain CDR1(SEQ ID NO: 145): 7,8 and 9 th bit

-light chain CDR2(SEQ ID NO: 153): 1,2, 3,4 and 5 th position

-light chain CDR3(seq id no165, 167,169,171,173, or 175): 4,5, 6 and 7 th bit

In certain embodiments, the substitution is a conservative substitution, as provided herein. In certain embodiments, one or more of the following substitutions or deletions may be made in any combination:

heavy chain CDR1(SEQ ID NO: 3): Y2F, H or S, A3T

Heavy chain CDR2(SEQ ID NO: 35): A1G, S3G, I, W or L, G4V, S, A or T, S5G or N, G6T or A, G7R, S, A, E or N, S8Y, L, R, I, N, Q, I or delete, T9 delete

Light chain CDR1(SEQ ID NO: 145): S7T, S8R or S9N

-light chain CDR2(SEQ ID NO: 153): Y1N, I or Q, G2V, A3G, S4T or Y, S5R, T or I

-light chain CDR3(seq id no165, 167,169,171,173, or 175): G4A, Q, N, L or H5I, L, V, Q, N or I6M, I7L

In addition, the antibody may further comprise one or more additions, deletions and/or substitutions in the framework region of one or more heavy or light chains as compared to the parent antibody. In one embodiment, at least one amino acid substitution in the at least one CDR contributes to an increase in binding affinity of the antibody as compared to its parent antibody. In another embodiment, the antibody has at least about 2-fold to about 10-fold greater affinity for FAP than the parent antibody (when comparing the antibody of the invention and the parent antibody in the same form, e.g., Fab form). In addition, antibodies derived from a parent antibody may incorporate any of the features described in the preceding paragraphs with respect to the antibodies of the invention, singly or in combination.

The invention also provides polynucleotides encoding antibodies that specifically bind FAP. In one aspect, the invention relates to an isolated polynucleotide encoding a polypeptide forming part of an anti-FAP antibody according to the invention described above. In one embodiment, the isolated polynucleotide encodes an antibody heavy chain and/or an antibody light chain that forms part of an anti-FAP antibody according to the invention described above.

In one embodiment, the invention relates to an isolated polynucleotide comprising a sequence encoding one or more (e.g., one, two, three, five, six, or four) of seq id nos3, 5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175, and 177 of a truncated form (or a light chain) or a light chain or light chain variant thereof containing a determinant (CDR) or a light chain or light chain variant thereof, as the CDR. In another embodiment, the polynucleotide comprises a light chain truncated version of a sequence selected from the group consisting of SEQ ID NOs3, 5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175 and 177, or a variant or truncated version or version of the above sequence (which variant or version contains at least three of the light chain truncated versions (e.g., HCDR1, e.g., HCDR 4682, GCR 4657, GCR) and/or light chain truncated versions (which comprise at least three of the sequence (e.g., HCDR 3982, GCR 2, e.g., HCDR2, GCR) and/or light chain truncated version of the above, as each of the three complementarity determining regions. In yet another embodiment, the polynucleotide comprises a sequence encoding three heavy chain CDRs (e.g., HCDR1, HCDR2, and HCDR 3) and three light chain (e.g., LCDR1, LCDR2, LCDR 4625, and LCDR 2) sequences selected from seq id nos3, 5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,169,171,173,175, and 177. In a particular embodiment, the polynucleotide encodes one or more CDRs comprising at least about one or more of the nucleotide sequences set forth in seq id nos4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 190, 189, 95, or 95% CDRs, A sequence that is 97%, 98%, 99%, or 100% identical.

In yet another embodiment, the polynucleotide comprises a heavy chain variable region encoding a sequence selected from SEQ ID NO:197, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:207, SEQ ID NO:211, SEQ ID NO:215, SEQ ID NO:219, SEQ ID NO:223, SEQ ID NO:227, SEQ ID NO:231, SEQ ID NO:235, SEQ ID NO:239, SEQ ID NO:243, SEQ ID NO:247, SEQ ID NO:251, SEQ ID NO:255, SEQ ID NO:259, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:271, SEQ ID NO:275, SEQ ID NO:279, SEQ ID NO:283, SEQ ID NO:287, SEQ ID NO:291, SEQ ID NO:295, SEQ ID NO:299, SEQ ID NO:303, SEQ ID NO:285, SEQ ID NO:311, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:199, SEQ ID NO:285, SEQ ID NO:225, SEQ ID NO:285, SEQ ID NO:269, SEQ ID NO:241, SEQ ID NO:225, SEQ ID NO:285, SEQ ID NO:241, SEQ ID NO:225, SEQ ID NO:285, SEQ ID NO:225, SEQ ID NO:241, SEQ ID NO:225, SEQ ID NO:281, SEQ ID NO:225, SEQ, Light chain variable region sequences of SEQ ID NO. 293, SEQ ID NO. 297, SEQ ID NO. 301, SEQ ID NO. 305, and SEQ ID NO. 309. In a particular embodiment, the polynucleotide encodes a heavy and/or light chain variable region comprising a sequence selected from the group of variable region nucleotide sequences set forth in seq id nos194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, and 312, or a combination thereof.

In a specific embodiment, the polynucleotide comprises a sequence encoding the heavy chain selected from the group consisting of SEQ ID NO:197, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:207, SEQ ID NO:211, SEQ ID NO:215, SEQ ID NO:219, SEQ ID NO:223, SEQ ID NO:227, SEQ ID NO:231, SEQ ID NO:235, SEQ ID NO:239, SEQ ID NO:243, SEQ ID NO:247, SEQ ID NO:251, SEQ ID NO:255, SEQ ID NO:259, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:271, SEQ ID NO:275, SEQ ID NO:279, SEQ ID NO:283, SEQ ID NO:287, SEQ ID NO:291, SEQ ID NO:295, SEQ ID NO:299, SEQ ID NO:303, SEQ ID NO:307, and SEQ ID NO:311 and a sequence encoding the constant region, in particular the human heavy chain. In a particular embodiment, the heavy chain constant region is a human IgG heavy chain constant region comprising an Fc region, in particular a human IgG1 heavy chain constant region. In a specific embodiment, the heavy chain constant region comprises the sequence SEQ ID NO 313. In another specific embodiment, the polynucleotide comprises a light chain sequence encoding a light chain region selected from the group consisting of SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:199, SEQ ID NO:205, SEQ ID NO:209, SEQ ID NO:213, SEQ ID NO:217, SEQ ID NO:221, SEQ ID NO:225, SEQ ID NO:229, SEQ ID NO:233, SEQ ID NO:237, SEQ ID NO:241, SEQ ID NO:245, SEQ ID NO:249, SEQ ID NO:253, SEQ ID NO:257, SEQ ID NO:261, SEQ ID NO:265, SEQ ID NO:269, SEQ ID NO:273, SEQ ID NO:277, SEQ ID NO:281, SEQ ID NO:285, SEQ ID NO:289, SEQ ID NO:293, SEQ ID NO:297, SEQ ID NO:301, SEQ ID NO:305, and SEQ ID NO:309 and a sequence encoding a light chain constant region, particularly a human light chain constant region. In a specific embodiment, the light chain constant region comprises the sequence SEQ ID NO 315.

In one embodiment, the invention relates to a composition comprising a first isolated polynucleotide encoding a polypeptide comprising at least the same amino acid sequence as a polynucleotide selected from the group consisting of SEQ ID NO197, SEQ ID NO 201, SEQ ID NO 203, SEQ ID NO 207, SEQ ID NO 211, SEQ ID NO 215, SEQ ID NO 219, SEQ ID NO 223, SEQ ID NO 227, SEQ ID NO 231, SEQ ID NO 235, SEQ ID NO 239, SEQ ID NO 243, SEQ ID NO 247, SEQ ID NO 251, SEQ ID NO 255, SEQ ID NO 259, SEQ ID NO 263, SEQ ID NO 267, SEQ ID NO 271, SEQ ID NO 275, SEQ ID NO 279, SEQ ID NO 283, SEQ ID NO 287, SEQ ID NO 291, SEQ ID NO 295, SEQ ID NO 299, SEQ ID NO 303, SEQ ID NO 307, SEQ ID NO193, SEQ ID NO 95, SEQ ID NO 99, SEQ ID NO 213, SEQ ID NO 195, SEQ ID NO 213, SEQ ID NO 195, SEQ ID NO 95, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265,269, 273, 277, 281, 285, 289, 293, 297, 301, 305, 309, 305, and 309.

In one embodiment, the invention relates to a composition comprising a first isolated polynucleotide comprising a sequence that is identical to or at least identical to a sequence selected from the group consisting of SEQ ID NO 198, SEQ ID NO 202, SEQ ID NO 204, SEQ ID NO 208, SEQ ID NO 212, SEQ ID NO 216, SEQ ID NO 220, SEQ ID NO 224, SEQ ID NO 228, SEQ ID NO 232, SEQ ID NO 236, SEQ ID NO 240, SEQ ID NO 244, SEQ ID NO 248, SEQ ID NO 252, SEQ ID NO 256, SEQ ID NO 260, SEQ ID NO 264, SEQ ID NO 268, SEQ ID NO 272, SEQ ID NO 276, SEQ ID NO 280, SEQ ID NO 284, SEQ ID NO 288, SEQ ID NO 292, SEQ ID NO 296, SEQ ID NO 300, SEQ ID NO 304, SEQ ID NO 308, SEQ ID NO 312, SEQ ID NO 95, SEQ ID NO 98, SEQ ID NO 194, SEQ ID NO 196, SEQ ID NO 218, SEQ ID NO 95, SEQ ID NO 25, SEQ ID NO, 226, 230, 234, 238, 242, 246, 250, 254, 258, 262, 266, 270, 274, 278, 282, 286, 290, 294, 298, 302, 306, and 310 sequences are at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

In a further aspect, the invention also relates to an isolated polypeptide encoded by any of the polynucleotides according to the invention described above.

In yet another aspect, an anti-FAP antibody according to any of the above embodiments may incorporate any of the features described in sections 1-6 below, singly or in combination:

1. affinity of antibody

In certain embodiments, an antibody provided herein has ≦ 1 μ M ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M) dissociation constant (K)_D). Preferably, the antibodies provided herein have a K of less than 1nM_DThe value (as determined by Surface Plasmon Resonance (SPR)) is combined into Fibroblast Activation Protein (FAP), in particular human FAP.

According to one embodiment, K is measured using surface plasmon resonance_D. Such assays may, for example, useThe T100 machine (GEHealthcare) was carried out at 25 ℃ with CM5 chips for antigen immobilization. Briefly, carboxymethylated dextran biosensor chips (CM5, ge healthcare) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. anti-His antibody (PentaHis, Qiagen) was diluted to 40. mu.g/ml with 10mM sodium acetate, pH5, followed by injection at a flow rate of 10. mu.l/min to achieve approximately 9000 Response Units (RU) of conjugated protein. After injection of anti-His antibody, 1M ethanolamine was injected to block unreacted groups. Subsequently, His-tagged antigen was injected at 10 μ l/min, 10mM20 sec (for Fab fragment measurement) or 20nM25 sec (for IgG antibody measurement), and captured via its His-tag by immobilized anti-His antibody. The protein and DNA sequences of suitable FAP protein constructs are shown in seq id no: 317-322. For kinetic measurements, serial dilutions of antibody were injected at 25 ℃ at a flow rate of 90 μ l/min in 10mM HEPES,150mM NaCl,3mM EDTA,0.05% surfactant P20, pH7.4 (Fab fragments were two-fold dilutions, ranging between 6.25nM and 200nM, or IgG was 5-fold dilution, ranging between 3.2pM and 10 nM). The following parameters were applied: the binding time was 180 seconds, the dissociation time was 300 seconds (for Fab) or 900 seconds (for Ig)G) Between each cycle, 60 seconds of 10mM glycine pH2 regeneration was performed. Using a simple one-to-one langmuir binding model (T100EvaluationSoftware) calculate the binding rate (k) by fitting the binding and dissociation sensorgrams simultaneously_on) And dissociation rate (k)_off). At a ratio of k_off/k_onCalculation of equilibrium dissociation constant (K)_D). See, e.g., Chen et al, J.mol.biol.293:865-881 (1999).

2. Antibody fragments

In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab '-SH, F (ab')₂Fv, and scFv fragments, and other fragments described below. For reviews of certain antibody fragments see Hudson et al Nat. Med.9: 129-.

For reviews of scFv fragments see, for example, Pl ü ckthun, eds (Springer-Verlag, New York), pp.269-315 (1994); also see WO93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458 for Fab and F (ab')₂See U.S. Pat. No.5,869,046 for a discussion of fragments.

Diabodies are antibody fragments with two antigen binding sites, which may be bivalent or bispecific. See, for example, EP404,097, WO1993/01161, Hudson et al, nat. Med.9: 129-. Tri-and tetrabodies are also described in Hudson et al, nat. Med.9: 129-.

Minibody refers to a bivalent, homodimeric scFv derivative containing the CH3 region of a constant region, typically an immunoglobulin, preferably an IgG, more preferably an IgG1, as a dimerization region. Typically, the constant region is linked to the scFv via a hinge region and/or linker region. Examples of minibody proteins can be found in Hu et al, cancer Res.56:3055-61 (1996).

Single domain antibodies are antibody fragments that comprise all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No.6,248,516B1).

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

3. Chimeric and humanized antibodies

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

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and FRs (or portions thereof) are derived from a human antibody sequence. Optionally, the humanized antibody will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanization can be achieved by a variety of methods, including but not limited to (a) grafting the entire non-human variable domain onto a human constant region to generate a chimeric antibody; (b) grafting only non-human (e.g., donor antibody) CDRs onto human (e.g., acceptor antibody) frameworks and constant regions with or without retention of critical framework residues (e.g., those important for retaining good antigen binding affinity or antibody function), (c) grafting only non-human specificity determining regions (SDRs or a-CDRs; residues critical for antibody-antigen interaction) onto human frameworks and constant regions, or (d) grafting entire non-human variable domains, but "masking" them with human-like moieties by replacing surface residues. Humanized antibodies and Methods for their production are reviewed, for example, in Almagro and Fransson, Front.biosci.13:1619-1633(2008), and further described, for example, in Riechmann et al, Nature332:323-329(1988), Queen et al, Proc.Nat' lAcad.Sci.USA86:10029-10033(1989), U.S. Pat. No.5,821,337,7,527,791,6,982,321 and 7,087,409, Jones et al, Nature321:522-525(1986), Morrison et al, Proc.Natl.Acad.Sci.81:6851-6855(1984), Morrison and Oi, Adv.munol.44: 65-92(1988), Verhoeyen et al, Science239:1534 (1988), Paslan et al, Patlan et al, 31: 31-32, CDR 169: 35-35 (CDR 33, 1994); padlan, mol.Immunol.28:489-498(1991) (describes "resurfacing"); dall' Acqua et al, Methods36:43-60(2005) (describing "FR shuffling"); and Osbourn et al, Methods36:61-68(2005) and Klimka et al, Br.J. cancer83:252-260(2000) (describing the "guided selection" method of FR shuffling).

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

4. Human antibodies

In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be generated using a variety of techniques known in the art. In general, human antibodies are described in vanDijk and vandeWinkel, curr. opin. pharmacol.5:368-74(2001), and Lonberg, curr. opin. immunol.20: 450-.

Human antibodies can be made by administering an immunogen to a transgenic animal that has been modified to produce fully human antibodies or fully antibodies with human variable regions in response to an antigenic challenge. Such animals typically contain all or part of a human immunoglobulin locus, which replaces an endogenous immunoglobulin locus, or which exists extrachromosomally or is randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus has typically been inactivated. For an overview of the method of obtaining human antibodies from transgenic animals, see Lonberg, nat. Biotech.23:1117-1125 (2005). See also, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE^TMA technique; U.S. Pat. No.5,770,429, which describesA technique; U.S. Pat. No.7,041,870, which describes K-MTechnology, and U.S. patent application publication No. US2007/0061900, which describesA technique). The human variable regions from the whole antibodies generated by such animals may be further modified, for example by combination with different human constant regions.

Human antibodies can also be generated by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, e.g., Kozbor J. Immunol.,133:3001(1984); Brodeur et al, monoclonal antibody production techniques and applications, pp 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al, J. Immunol.,147:86 (1991)). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al, Proc.Natl.Acad.Sci.USA,103:3557-3562 (2006). Other methods include those described, for example, in U.S. Pat. No.7,189,826, which describes the production of monoclonal human IgM antibodies from hybridoma cell lines, and Ni, Xiandai Mianyixue,26(4):265-268(2006), which describes human-human hybridomas. The human hybridoma technique (Trioma technique) is also described in Vollmers and Brandlein, Histologyand Histopapathology, 20(3): 927-.

Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

5. Library-derived antibodies

Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies having a desired activity or activities. For example, various methods for generating phage display libraries and screening such libraries for antibodies possessing desired binding characteristics are known in the art. Such methods are reviewed, for example, in Hoogenboom et al, Methodesinmolecular biology178:1-37 (O' Brien et al, eds., HumanPress, Totowa, NJ,2001), and further described, for example, in McCafferty et al, Nature348:552-554, Clackson et al, Nature352:624-628(1991), Marks et al, J.Mol.biol.222:581-597(1992), Marks and Bradbury in MethodesinMolecular biology248:161-175(Lo et al, HumanPress, Totowa, NJ,2003), Sidhu et al, J.mol.338 (2): 299-2004 (2004); Lee et al, J.mol.340 (5):1073 (Acetc. 12419, Nature: 119-72, USA) and in Legend et al, Nature # 72 (USA) in Legend).

In some phage display methods, the repertoire of VH and VL genes, respectively, is cloned by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phages, as described in Winter et al, Ann. Rev. Immunol.,12:433-455 (1994). Phage typically display antibody fragments either as single chain fv (scfv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the natural repertoire can be cloned (e.g., from humans) to provide a single source of antibodies to a large panel of non-self and also self-antigens in the absence of any immunization, as described by Griffiths et al, EMBOJ,12: 725-. Finally, non-rearranged V gene segments can also be synthesized by cloning non-rearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and effecting rearrangement in vitro, as described by Hoogenboom and Winter, J.mol.biol.,227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No.5,750,373, and U.S. patent publication Nos. 2005/0079574,2005/0119455,2005/0266000,2007/0117126,2007/0160598,2007/0237764,2007/0292936 and 2009/0002360.

Antibodies or antibody fragments isolated from a human antibody library are considered to be human antibodies or human antibody fragments herein.

6. Multispecific antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for FAP and the other is for any other antigen. In certain embodiments, a bispecific antibody may bind to two different epitopes of FAP. Bispecific antibodies may also be used to localize cytotoxic agents to FAP-expressing cells. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature305:537 (1983)), WO93/08829, and Traunecker et al, EMBO J.10:3655 (1991)), and "bump-in-hole" engineering (see, e.g., U.S. Pat. No.5,731,168). Effects can also be manipulated electrostatically by engineering for the generation of antibody Fc-heterodimer molecules (WO2009/089004a 1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No.4,676,980, and Brennan et al, Science,229:81 (1985)); the use of leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al, J.Immunol.,148(5):1547-1553 (1992)); the "diabody" technique used to generate bispecific antibody fragments is used (see, e.g., Hollinger et al, Proc. Natl. Acad. Sci. USA,90: 6444-; and the use of single chain fv (scFv) dimers (see, e.g., Gruber et al, J.Immunol.,152:5368 (1994)); and making a trispecific antibody to generate a multispecific antibody as described, for example, in Tutt et al j.

Also included herein are engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US2006/0025576a 1).

Antibodies or fragments herein also include "dual action fabs" or "DAFs" comprising an antigen binding site that binds FAP and another, different antigen (see, e.g., US 2008/0069820).

7. Antibody variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are encompassed. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics, e.g., antigen binding.

a)Substitution, insertion, and deletion variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs and FRs.

Amino acid substitutions can result in the substitution of one amino acid with another having similar structural and/or chemical properties, e.g., conservative amino acid substitutions. "conservative" amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, phenylalanine, tryptophan, and methionine; polar neutral amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Conservative substitutions are shown in table 2 under the heading of "preferred substitutions". More substantial variations are provided in table 2 under the heading of "exemplary substitutions" and are described further below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the product screened for a desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

TABLE 2

According to common side chain properties, amino acids can be grouped as follows:

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

(2) neutral, hydrophilic: cys, Ser, Thr, Asn, Gln;

(3) acidic: asp, Glu;

(4) basic: his, Lys, Arg;

(5) residues that influence chain orientation: gly, Pro;

(6) aromatic: trp, Tyr, Phe.

Non-conservative substitutions may entail replacing one of these classes with a member of the other class. For example, an amino acid substitution can also result in the substitution of one amino acid with another amino acid having a different structural and/or chemical property, e.g., substitution of one amino acid from one group (e.g., polar) with another amino acid from a different group (e.g., basic). The variation allowed can be determined experimentally by using recombinant DNA techniques to systematically generate amino acid insertions, deletions, or substitutions in a polypeptide molecule and assaying the resulting recombinant variants for activity.

One class of surrogate variants involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study will have an alteration (e.g., an improvement) in certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and/or will substantially retain certain biological properties of the parent antibody. Exemplary surrogate variants are affinity matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

Changes (e.g., substitutions) can be made to HVRs, for example, to improve antibody affinity. Such changes can be made to HVR "hot spots", i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, methods mol. biol.207: 179. 196 (2008)), and/or SDRs (a-CDRs), where the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction and re-selection of secondary libraries has been described, for example, in Hoogenboom, et al, methods molecular biology178:1-37 (O' Brien et al, eds., HumanPress, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Then, a secondary library is created. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-directed method in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR 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 frequently targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) may be made to HVRs that do not substantially reduce binding affinity. Such changes may be outside of HVR "hotspots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unaltered or contains no more than 1,2 or 3 amino acid substitutions.

One method that can be used to identify residues or regions of an antibody that can 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 residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with a neutral or negatively charged amino acid (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 indicate functional sensitivity to the initial substitution. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify contact points between the antibody and the antigen. As alternative candidates, such contact and adjacent residues may be targeted or eliminated. Variants can be screened to determine if they contain the desired property.

Amino acid sequence insertions include amino and/or carboxy-terminal fusions ranging in length from 1 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 the antibody molecule include fusions of the N-or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a polypeptide that extends the serum half-life of the antibody.

b)Glycosylation variants

In some embodiments, the oligosaccharides in the antibodies of the invention may be modified to create antibody variants with certain improved properties.

In one aspect, the invention provides glycoforms of anti-FAP antibodies with increased effector function, including antibody-dependent cellular cytotoxicity. Glycosylation engineering of antibodies has been previously described. See, for example, U.S. Pat. No.6,602,684, which is incorporated herein in its entirety by reference. Methods of producing anti-FAP antibodies from host cells having altered activity of genes involved in glycosylation are also described in detail herein (see, e.g., the preceding section entitled "recombinant methods and compositions").

IgG molecules carry two N-linked oligosaccharides in their Fc region, one on each heavy chain. As with any glycoprotein, antibodies are produced in populations of glycoforms sharing the same polypeptide backbone, but having different oligosaccharides attached to glycosylation sites. Oligosaccharides commonly found in the Fc region of serum IgG are complex biantennary (Wormald et al, Biochemistry36:130-38(1997), with low levels of terminal sialic acid and bipartite N-acetylglucosamine (GlcNAc), and variable degrees of terminal galactosylation and core fucosylation (fucose attached to GlcNAc residues in the "backbone" of the biantennary oligosaccharide structure.) some studies suggest that the minimum carbohydrate structure required for Fc γ R binding is located within the oligosaccharide core. Lund et al, J.Immunol.157:4963-69 (1996)).

Mouse or hamster derived cell lines used in industry and academia to produce antibodies typically attach desired oligosaccharide determinants to the Fc site. However, IgG expressed in these cell lines lacks bisecting GlcNAc found in lower amounts in serum IgG. Lifely et al, Glycobiology318:813-22 (1995). In the N-linked glycosylation pathway, bisecting GlcNAc is added by GnTIII. Schachter, biochem. CellBiol.64:163-81 (1986).

Et al used a single antibody to generate CHO cell lines previously engineered to express different levels of cloned GnTIII enzyme genes in an externally regulated manner: (P, et al, Nature Biotechnol.17:176-180 (1999)). This approach establishes for the first time a strict correlation between glycosyltransferase (e.g., GnTIII) expression and ADCC activity of the modified antibody. As such, the invention encompasses anti-FAP antibodies comprising an Fc region or a region equivalent to an Fc region having altered glycosylation resulting from altered expression levels of glycosyltransferase genes in an antibody-producing host cell. In a specific embodiment, the change in the level of gene expression is an increase in GnTIII activity. Increased GnTIII activity results in an increased percentage of bisected oligosaccharides and a decreased percentage of fucosylated oligosaccharides in the Fc region of the antibody. The antibody or fragment thereof has increased Fc receptor binding affinity and increased effector function.

Antibodies with bisected oligosaccharides, e.g. are providedWherein the biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003/011878 (Jean-Mairet et al); U.S. Pat. No.6,602,684(Etc.); and US2005/0123546 (Etc.).

In one embodiment, the anti-FAP antibody of the invention has an increased proportion of bisected oligosaccharides in the Fc region due to its oligosaccharide modification by the methods of the invention. In one embodiment, the percentage of bisected N-linked oligosaccharides in the Fc region of an anti-FAP antibody of the invention is at least about 10% to about 100%, specifically at least about 50%, more specifically at least about 60%, at least about 70%, at least about 80%, or at least about 90-95% of the total oligosaccharides. The bisected oligosaccharides may be of the hybrid or complex type.

In another embodiment, the anti-FAP antibody of the invention has an increased proportion of non-fucosylated oligosaccharides in the Fc region as a result of its oligosaccharide modification by the method of the invention. In one embodiment, the percentage of nonfucosylated oligosaccharides is at least about 20% to about 100%, particularly at least about 50%, at least about 60% to about 70%, and more particularly at least about 75%. The nonfucosylated oligosaccharides may be of the hybrid or complex type.

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. complexed, heterozygous and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g. as described in WO 2008/077546. Asn297 refers to the asparagine residue located 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. The relative amount of fucose refers to the percentage of fucose-containing structures relative to all sugar structures (e.g., complex, heterozygous and high mannose structures) identified in the N-glycosidase F-treated sample according to MALDI-TOFMS. Such fucosylated variants may have improved ADCC function.

Glycoengineering methods useful for anti-FAP antibodies of the invention have been described in great detail in U.S. Pat. nos. 6,602,684; U.S. patent application publication nos. 2004/0241817a 1; U.S. patent application publication nos. 2003/0175884a 1; U.S. provisional patent application No.60/441,307 and WO2004/065540, the entire contents of each of which are hereby incorporated by reference in their entirety. Alternatively, the compositions may be prepared according to U.S. patent application publication No.2003/0157108(Genentech) or EP1176195a 1; WO 03/084570; WO03/085119 and U.S. patent application publication No. 2003/0115614; no. 2004/093621; no. 2004/110282; no. 2004/110704; no. 2004/132140; niwaet al, JImmunol methods306,151/160 (2006); the anti-FAP antibodies of the invention can be glycoengineered to have reduced fucose residues in the Fc region by techniques disclosed in U.S. patent No.6,946,292 (Kyowa). Glycoengineered anti-FAP antibodies of the invention may also be produced in expression systems that produce modified glycoproteins, such as those taught in chinese patent application publication nos. 60/344,169 and WO03/056914(GlycoFi, Inc.) or WO2004/057002 and WO2004/024927 (Greenovation).

Other examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: WO2000/61739, WO2001/29246, US2002/0164328, US2004/0109865, WO2005/035586, WO2005/035778, WO2005/053742, WO2002/031140, Okazaki et al J.mol.biol.336:1239-1249(2004), Yamane-Ohnuki et al Biotech.Bioeng.87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation deficient Lec13CHO cells (Ripka et al Arch. biochem. Biophys.249:533-545(1986); U.S. patent application No. US2003/0157108A1, Presta, L; and WO2004/056312A1, Adams et al, inter alia, in example 11), and knock-out cell lines such as alpha-1, 6-fucosyltransferase gene FUT8 knock-out 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).

In a specific embodiment, the anti-FAP antibody of the invention has a dichotomized, nonfucosylated oligosaccharide with an increased proportion of Fc regions. The bisected, nonfucosylated oligosaccharides may be hybrids or complexes. In particular, the methods of the invention can be used to generate anti-FAP antibodies in which at least about 10% to about 100%, specifically at least about 15%, more specifically at least about 20% to about 25%, and more specifically at least about 30% to about 35% of the oligosaccharides in the Fc region of the antigen binding molecule are bisected, nonfucosylated. The anti-FAP antibodies of the invention may also comprise an Fc region wherein at least about 10% to about 100%, specifically at least about 15%, more specifically at least about 20% to about 25%, and more specifically at least about 30% to about 35% of the oligosaccharides in the Fc region of the antibody are bisected, hybrid, nonfucosylated.

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of glycosylation of the antibody. Addition or deletion of glycosylation sites of an antibody can be conveniently achieved by altering the amino acid sequence such that one or more glycosylation sites are created or eliminated.

Antibody variants having at least one galactose residue in an oligosaccharide attached to an Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997/30087 (Patel et al); WO1998/58964(Raju, S.); and WO1999/22764(Raju, S.).

The increase in ADCC or other effector function of the anti-FAP antibodies of the invention is also achieved by increasing the affinity of the antigen-binding molecule for FAP, for example by affinity maturation or other methods of improving affinity (see Tang et al, J.Immunol.2007,179: 2815-2823) or by amino acid modification in the Fc region (as described below). The present invention also covers combinations of these approaches.

c)Fc region variants

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

In certain embodiments, the invention encompasses antibody variants possessing some, but not all, effector functions that make them desirable candidates for applications where the in vivo half-life of the antibody is important, while certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays may be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that the antibody lacks fcyr binding (and therefore potentially lacks ADCC activity), but retains FcRn binding ability. The major cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of ravatch and Kinet, Annu. Rev. Immunol.9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of molecules of interest are described in U.S. Pat. No.5,500,362 (see, e.g., Hellstrom, I.e., Proc. Nat' l Acad. Sci. USA83: 7059-. Alternatively, non-radioactive methods can be employed (see, e.g., ACTI for flow cytometry)^TMNon-radioactive cytotoxicity assays (Celltechnology, Inc. mountain View, CA; and CytoTox)Non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively/additionally, the ADCC activity of a molecule of interest may be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al Proc. Nat' lAcad. Sci. USA95:652-1998) In (1). A C1q binding assay may also be performed to confirm that the antibody is unable to bind C1q, and therefore lacks CDC activity. See, e.g., the C1q and C3C binding ELISAs in WO2006/029879 and WO 2005/100402. To assess complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al, J.Immunol. methods202:163(1996); Cragg, M.S. et al, Blood101: 1045-. FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, e.g., Petkova, s.b. et al, Int' l.immunol.18(12): 1759-.

Antibodies with reduced effector function include those having substitutions in one or more of residues 238,265,269,270,297,327 and 329 of the Fc region (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 having substitutions of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or reduced binding to FcR are described (see, e.g., U.S. Pat. No.6,737,056; WO2004/056312, and Shields et al, J.biol. chem.9(2):6591-6604 (2001)).

In certain embodiments, an 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.

In some embodiments, alterations are made to the Fc region that result in altered (i.e., improved or reduced) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No.6,194,551, WO99/51642, and Idusogene et al J.Immunol.164: 4178-.

Antibodies with extended half-life and improved binding to neonatal Fc receptor (FcRn) responsible for the transfer of maternal IgG to the fetus are described in US2005/0014934A1(Hinton et al), the neonatal Fc receptor (FcRn) and are responsible for the transfer of maternal IgG to the fetus (Guyer et al, J.Immunol.117:587(1976) and Kim et al, J.Immunol.24:249 (1994)). Those antibodies comprise an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of residues 238,256,265,272,286,303,305,307,311,312,317,340,356,360,362,376,378,380,382,413,424 or 434 of the Fc region, for example, at residue 434 of the Fc region (U.S. patent No.7,371,826).

As to other examples concerning Fc region variants, see also U.S. patent application No.60/439,498; 60/456,041, respectively; 60/514,549, respectively; or WO2004/063351 (variant Fc regions with increased binding affinity due to amino acid modifications); or U.S. patent application No.10/672,280 or WO2004/099249 (Fc variants with altered binding to fcyr due to amino acid modifications); duncan and Winter, Nature322:738-40 (1988); U.S. Pat. Nos. 5,648,260; U.S. Pat. Nos. 5,624,821; and WO 94/29351.

d)Cysteine engineered antibody variants

In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g., "thiomabs," in which one or more residues of the antibody are replaced with cysteine residues. In particular embodiments, the substituted residues are present at accessible sites of the antibody. By replacing those residues with cysteine, the reactive thiol groups are thereby localized to accessible sites of the antibody and can be used to conjugate the antibody with other moieties, such as drug moieties or linker-drug moieties, to create antibody conjugates, as further described herein. In certain embodiments, cysteine may be substituted for any one or more of the following residues: v205 of the light chain (Kabat numbering); a118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering). Cysteine engineered antibodies can be produced as described, for example, in U.S. patent No.7,521,541.

e)Antibody derivatives

In certain embodiments, the terms provided herein may be further modifiedThe antibodies provided may contain additional non-proteinaceous moieties known in the art and readily available. Suitable moieties for derivatization of the antibody include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trisAlkanes, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative will be used for therapy under specified conditions, and the like.

In another embodiment, conjugates of an antibody and a non-proteinaceous moiety that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al, Proc. Natl. Acad. Sci. USA102: 11600-. The radiation can be of any wavelength and includes, but is not limited to, wavelengths that are not damaging to normal cells, but heat the non-proteinaceous moiety to a temperature at which cells in the vicinity of the antibody-non-proteinaceous moiety are killed.

B. Recombinant methods and compositions

Recombinant methods and compositions can be used to generate antibodies, for example, as described in U.S. Pat. No.4,816,567. In one embodiment, an isolated polynucleotide encoding an anti-FAP antibody described herein is provided. Such polynucleotides may encode an amino acid sequence comprising a VL of an antibody and/or an amino acid sequence comprising a VH (e.g., the light and/or heavy chain of an antibody). In yet another embodiment, one or more vectors (e.g., cloning vectors or expression vectors) comprising such polynucleotides are provided. In yet another embodiment, host cells comprising such polynucleotides or such vectors are provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a polynucleotide encoding an amino acid sequence comprising a VL of an antibody and an amino acid sequence comprising a VH of an antibody (e.g., a polycistronic vector), or (2) a first vector comprising a polynucleotide encoding an amino acid sequence comprising a VL of an antibody and a second vector comprising a polynucleotide encoding an amino acid sequence comprising a VH of an antibody. In one embodiment, the host cell is a eukaryotic cell, particularly a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, Baby Hamster Kidney (BHK) cell, or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of producing an anti-FAP antibody is provided, wherein the method comprises culturing a host cell comprising a polynucleotide encoding the antibody under conditions suitable for expression of the antibody, as provided above, and optionally, recovering the antibody from the host cell (or host cell culture broth).

For recombinant production of anti-FAP antibodies, one or more polynucleotides encoding the antibodies (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence for the anti-FAP antibody together with appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination/genetic recombination. See, for example, techniques described in Maniatis et al, Molecularcloning, ALABORATORYMANUAL, ColdSpringHarbor laboratory, N.Y. (1989), and Ausubel et al, CurentPROTOCOLS IN MOLECULARBIOLOGY, Greene publishing Association and Wiley Interscience, N.Y. (1989).

In one embodiment, one or several polynucleotides encoding anti-FAP antibodies may be expressed under the control of a constitutive promoter or alternatively a regulated expression system. Suitable regulatory expression systems include, but are not limited to, tetracycline-regulated expression systems, ecdysone inducible expression systems, lac switch expression systems, glucocorticoid inducible expression systems, temperature inducible promoter systems, and metallothionein metal inducible expression systems. If several different polynucleotides encoding the antibodies of the invention are contained within a host cell system, some of them may be expressed under the control of a constitutive promoter, while others are expressed under the control of a regulatory promoter.

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,5,789,199 and 5,840,523 (see also Charlton, methods molecular biology, Vol.248 (compiled by B.K.C.Lo., HumanaPress, Totowa, NJ,2003), pp.245-254, which describes expression of antibody fragments in E.coli (E.coli)). After expression, the antibody can be isolated from the bacterial cell mass paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized" resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, nat. Biotech.22: 1409-. Such expression systems are also taught in U.S. patent application No.60/344,169 and WO03/056914 (methods for producing human-like glycoproteins in non-human eukaryotic host cells).

Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified which can be used with insect cells, particularly for transfecting spodoptera frugiperda (spodoptera frugiperda) cells.

Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177,6,040,498,6,420,548,7,125,978 and 6,417,429 (which describe PLANTIBODIIES for antibody production in transgenic plants^TMA technique).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (293 or 293T cells as described, e.g., in Graham et al, J.GenVirol.36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli (sertoli) cells (TM 4 cells, as described, for example, in Mather, biol. reprod.23:243-251 (1980)); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); bovine rat (buffaloat) hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (HepG 2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather et al, AnnalsN.Y.Acad.Sci.383:44-68 (1982); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp 2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, methods in molecular biology, Vol.248 (B.K.C.Lo eds., HumanaPress, Totowa, NJ), pp.255-268 (2003).

Generally, stable expression is preferred for transient expression, as it generally achieves more reproducible results, and is also more suitable for large-scale production; however, it is within the skill of the person skilled in the art to determine whether transient expression is better for a particular situation.

The invention further relates to a method for modifying the glycosylation profile of an anti-FAP antibody of the invention produced by a host cell, comprising expressing in said host cell one or more polynucleotides encoding an anti-FAP antibody and one or more polynucleotides encoding a polypeptide having glycosyltransferase activity, or a vector comprising such polynucleotides. In general, any type of cultured cell line, including those discussed above, may be used to generate cell lines for the production of anti-FAP antibodies with altered glycosylation patterns. Preferred cell lines include CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER. c6 cells or hybridoma cells, and other mammalian cells. Polypeptides having glycosyltransferase activity include β (1,4) -N-acetylglucosaminyltransferase III (GnTIII), α -mannosidase II (ManII), β (1,4) -galactosyltransferase (GalT), β (1,2) -N-acetylglucosaminyltransferase I (GnTI), and β (1,2) -N-acetylglucosaminyltransferase II (GnTII). In one embodiment, a combination of polynucleotides encoding polypeptides having glycosyltransferase activity (e.g., GnTIII and ManII) is expressed in a host cell. Likewise, the methods also encompass expressing one or more polynucleotides encoding anti-FAP antibodies in a host cell in which the glycosyltransferase gene has been disrupted or otherwise inactivated (e.g., a host cell in which the activity of the gene encoding an α 1,6 core fucosyltransferase has been knocked out). In a particular embodiment, the anti-FAP antibodies of the invention may be produced in a host cell that further expresses a polynucleotide encoding a polypeptide having GnTIII activity to modify the glycosylation pattern of the antibody described below. In a particular embodiment, the polypeptide having GnTIII activity is a fusion polypeptide comprising the golgi localization domain of a golgi resident polypeptide. In another specific embodiment, expression of an anti-FAP antibody of the invention in a host cell expressing a polynucleotide encoding a polypeptide having GnTIII activity produces an anti-FAP antibody with increased Fc receptor binding affinity and/or increased effector function. Thus, in one embodiment, the present invention relates to a host cell comprising (a) one or more isolated polynucleotides comprising a sequence encoding a polypeptide having GnTIII activity; and (b) one or more isolated polynucleotides encoding an anti-FAP antibody of the invention. In a specific embodiment, the polypeptide having GnTIII activity is a fusion polypeptide comprising the catalytic domain of GnTIII and the golgi localization domain of a heterologous golgi resident polypeptide. In particular, the golgi localization domain is the golgi localization domain of mannosidase II. Methods for generating such fusion polypeptides and using them to generate antibodies with increased effector function are disclosed in WO 2004/065540; U.S. provisional patent application No.60/495,142 and U.S. patent application publication No.2004/0241817, the entire contents of which are expressly incorporated herein by reference. In another embodiment, the host cell further comprises an isolated polynucleotide comprising a sequence encoding a polypeptide having mannosidase ii (manii) activity. Polynucleotides encoding polypeptides, like polynucleotides encoding anti-FAP antibodies, may be expressed under the control of a constitutive promoter or alternatively a regulated expression system. Such systems are well known in the art and include the systems discussed above.

A host cell containing the coding sequence for an anti-FAP antibody and/or the coding sequence for a polypeptide having glycosyltransferase activity and expressing a biologically active gene product can be identified, for example, by: DNA-DNA or DNA-RNA hybridization; the presence or absence of "marker" gene function; assessing the level of transcription, as measured by expression of the respective mRNA transcript in the host cell; and detecting the gene product, as measured by immunoassay or by its biological activity (methods well known in the art). GnTIII or ManII activity can be detected, for example, by using lectins that bind to the biosynthesis products of GnTIII or ManII. An example of such a lectin is the E4-PHA lectin, which preferentially binds bisected GlcNAc-containing oligosaccharides. Biosynthetic products of polypeptides having GnTIII or ManII activity (i.e., specific oligosaccharide structures) can also be detected by mass spectrometry analysis of oligosaccharides released from glycoproteins produced by cells expressing the polypeptides. Alternatively, a functional assay that measures increased effector function or increased Fc receptor binding mediated by antibodies produced by cells engineered with a polynucleotide encoding a polypeptide having GnTIII activity may be used.

The present invention also relates to a method for producing an anti-FAP antibody with modified oligosaccharides, comprising (a) culturing a host cell engineered to express at least one polynucleotide encoding a polypeptide having glycosyltransferase activity under conditions permissive for production of an anti-FAP antibody according to the present invention, wherein the polypeptide having glycosyltransferase activity is expressed in an amount sufficient to modify oligosaccharides in the Fc region of the anti-FAP antibody produced by the host cell; and (b) isolating the anti-FAP antibody. In one embodiment, the polypeptide having glycosyltransferase activity is GnTIII. In another embodiment, there are two polypeptides having glycosyltransferase activity. In a specific embodiment, the two peptides having glycosyltransferase activity are GnTIII and ManII. In another embodiment, the polypeptide having glycosyltransferase activity is a fusion polypeptide comprising the catalytic domain of GnTIII. In a more specific embodiment, the fusion polypeptide further comprises a golgi localization domain of a golgi resident polypeptide. In particular, the golgi localization domain is the localization domain of mannosidase II or GnTI, most particularly the localization domain of mannosidase II. Alternatively, the golgi localization domain is selected from the group consisting of: the localization domain of mannosidase I, the localization domain of GnTII, and the localization domain of alpha 1,6 core fucosyltransferase.

In a specific embodiment, the modified anti-FAP antibody produced by the method or host cell described above has an IgG constant region comprising an Fc region, or a fragment thereof. In another specific embodiment, the anti-FAP antibody is a humanized or human antibody or fragment thereof comprising an Fc region.

anti-FAP antibodies with altered glycosylation produced by the methods or host cells described above typically exhibit increased Fc receptor binding affinity and/or increased effector function due to modification of the host cell (e.g., by expression of a glycosyltransferase gene). Preferably, the increased Fc receptor binding affinity is increased binding to an Fc γ activating receptor, most preferably an Fc γ RIIIa receptor. Preferably, the increased effector function is an increase in one or more of: increased antibody-dependent cell cytotoxicity, increased antibody-dependent cellular phagocytosis (ADCP), increased cytokine secretion, increased immune complex-mediated antigen uptake by antigen presenting cells, increased Fc-mediated cellular cytotoxicity, increased binding to NK cells, increased binding to macrophages, increased binding to polymorphonuclear cells (PMNC), increased binding to monocytes, increased cross-linking of target-bound antibodies, increased apoptosis-inducing direct signaling, increased dendritic cell maturation, and increased T-cell priming.

C. Assay method

The anti-FAP antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.

1. Binding assays and other assays

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

In another aspect, a competition assay can be used to identify an antibody that competes for binding to FAP with another specific anti-FAP antibody. In certain embodiments, such competitive antibodies bind to the same epitope (e.g., a linear or conformational epitope) as the epitope bound by the other specific anti-FAP antibody. A detailed exemplary method for locating epitopes bound by antibodies is described in Morris (1996) "epitome mapping protocols", Methodsinmolecular biology vol.66(HumanaPress, Totowa, N.J.).

In one exemplary competition assay, immobilized FAP is incubated in a solution comprising a first labeled antibody that binds FAP, e.g., the 3F2 antibody described in the examples, and a second unlabeled antibody that is to be tested for the ability to compete with the first antibody for binding to FAP. The second antibody may be present in the hybridoma supernatant. As a control, immobilized FAP was incubated in a solution comprising the first labeled antibody but no second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to FAP, excess unbound antibody is removed and the amount of label associated with the immobilized FAP is measured. If the amount of label associated with immobilized FAP in the test sample is substantially reduced compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to FAP. See Harlowland (1988) Antibodies, ALaboratoryManuatch.14 (ColdSpringHarbor laboratory, ColdSpringHarbor, NY).

2. Activity assay

In one aspect, assays for identifying anti-FAP antibodies having biological activity are provided. Biological activity may include, for example, lysis of targeted cells, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or induction of apoptosis. Antibodies having such biological activity in vivo and/or in vitro are also provided. In certain embodiments, antibodies of the invention are tested for such biological activity.

An "exemplary assay for testing ADCC" is described above (see "definitions" below: "antibody with elevated ADCC") and in example 11. Assays for detecting cell lysis (e.g., by measuring LDH release) or apoptosis (e.g., using TUNEL assay) are well known in the art. Assays for measuring ADCC or CDC are also described in WO2004/065540 (see example 1 therein), the entire contents of which are incorporated herein by reference.

D. Antibody conjugates

The invention also provides conjugates comprising an anti-FAP antibody herein conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope.

In one embodiment, in an antibody-drug conjugate (ADC), the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. nos. 5,208,020, 5,416,064 and european patent EP0425235B 1); auristatins such as monomethyl auristatin drug modules DE and DF (MMAE and MMAF) (see U.S. Pat. nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin (dolastatin); calicheamicin (calicheamicin) or a derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586,5,739,116,5,767,285,5,770,701,5,770,710,5,773,001 and 5,877,296; Hinman et al, cancer Res.53: 3336-; anthracyclines such as daunomycin (daunomycin) or doxorubicin (doxorubicin) (see Kratz et al, Current Med. chem.13: 477-; methotrexate; vindesine (vindesine); taxanes (taxanes) such as docetaxel (docetaxel), paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes (trichothecenes); and CC 1065.

In another embodiment, an antibody conjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, the enzymatically active toxins include, but are not limited to diphtheria a chain, non-binding active fragments of diphtheria toxin, exotoxin a chain (from pseudomonas aeruginosa), ricin a chain, abrin a chain, modeccin a chain, α -sarcin (sarcin), aleurites fordii (aleutis fordii) toxic protein, dianthus chinensis (dianthin) toxic protein, phytolacca americana (phytolacacaricanaria) protein (PAPI, PAPII and PAP), momordica charantia (momocarratia) inhibitor, curculin (curcin), crotin (crotin), saponaria officinalis (sapaonaria officinalis) inhibitor, gelonin (gelonin), mitomycin (mitrellin), tricin (curcin), curculin (trichothecin), enomycin (trichothecin), and trichothecin (trichothecin).

In another embodiment, the antibody conjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for use in generating radioconjugates. Examples include At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²And radioactive isotopes of Lu. Where a radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, for example tc99m or I123, or a spin label for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

A variety of bifunctional protein coupling agents may be used to generate conjugates of the antibody and cytotoxic agent, such as N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), imidoesters (such as dimethyl adipimidate hcl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) -ethylenediamine), diisothiocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene) is used. For example, a ricin immunotoxin may be prepared as described in Vitetta et al, Science238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies. See WO 94/11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al, cancer Res52: 127-.

Conjugates herein expressly encompass, but are not limited to, such conjugates prepared with crosslinking agents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl- (4-vinylsulfone) benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A.).

E. Methods and compositions for diagnosis and detection

In certain embodiments, any of the anti-FAP antibodies provided herein can be used to detect the presence of FAP in a biological sample. As used herein, the term "detecting" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as cells or tissues from brain, breast/breast, colon, kidney, liver, lung, ovary, pancreas, prostate, skeletal muscle, skin, small intestine, stomach, or uterus, and also cell or tissue tumors of these organs.

In one embodiment, anti-FAP antibodies for use in a diagnostic or detection method are provided. In yet another aspect, a method of detecting the presence of FAP in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample (optionally with a control sample) with an anti-FAP antibody, as described herein, under conditions that allow the anti-FAP antibody to bind to FAP, and detecting whether a complex is formed between the anti-FAP antibody and FAP. Such methods may be in vitro or in vivo. In one embodiment, an anti-FAP antibody is used to select a subject suitable for treatment with an anti-FAP antibody, e.g., wherein FAP is a biomarker for selecting patients.

Exemplary disorders that can be diagnosed using the antibodies of the invention include diseases associated with FAP expression, such as cancer and certain inflammatory conditions.

In one aspect, there is provided a method of diagnosing a disease in a subject, the method comprising administering to the subject an effective amount of a diagnostic agent, wherein the diagnostic agent comprises an anti-FAP antibody described herein and a label, typically an imaging agent, that allows for detection of a complex of the diagnostic agent and FAP.

In certain embodiments, labeled anti-FAP antibodies are provided. Labels include, but are not limited to, labels or moieties that are directly detectable (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and moieties that are indirectly detectable, such as enzymes or ligands, for example, via enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes³²P、¹⁴C、¹²⁵I、³H. And¹³¹I. fluorophores such as rare earth chelates or luciferin and derivatives thereof, rhodamine (rhodamine) and derivatives thereof, dansyl, umbelliferone, luciferases, e.g., firefly and bacterial luciferases (U.S. Pat. No.4,737,456), luciferin, 2, 3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase (which are coupled to an enzyme employing a hydrogen peroxide oxidation dye precursor such as HRP), lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, phage labels, stable free radicals, and the like.

F. Pharmaceutical formulations

Pharmaceutical formulations of the anti-FAP antibody as described herein are prepared by mixing such antibodies of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's pharmaceutical sciences 16 th edition, Osol, a. eds. (1980)) in a lyophilized formulation or as an aqueous solution. Generally, pharmaceutically acceptable carriers are used at the dosages and in amounts indicatedConcentrations are non-toxic to recipients and include, but are not limited to, buffers such as phosphate, citrate, acetate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; hydrocarbyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further comprise an interstitial drug dispersant such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (r: (r) ())Baxter International, Inc.). Certain exemplary shasegps and methods of use, including rHuPH20, are described in U.S. patent publication nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

An exemplary lyophilized antibody formulation is described in U.S. Pat. No.6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulation comprising a histidine-acetate buffer.

The formulations herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably those compounds whose activities are complementary and do not adversely affect each other. For example, if the disease to be treated is cancer, it may be desirable to further provide one or more anti-cancer agents, such as chemotherapeutic agents, inhibitors of tumor cell proliferation, or activators of tumor cell apoptosis. Such active components are suitably present in combination in amounts effective for the desired purpose.

The active ingredient may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed, for example, in Remington's pharmaceutical sciences, 16 th edition, Osol, A. eds (1980).

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

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

The molecules described herein can be in a variety of dosage forms including, but not limited to, liquid solutions or suspensions, tablets, pills, powders, suppositories, polymeric microcapsules or microbubbles, liposomes, and injectable or infusible solutions. The preferred form depends on the mode of administration and therapeutic application, but will typically be an injectable or infusible solution.

G. Therapeutic methods and compositions

Any of the anti-FAP antibodies provided herein or pharmaceutical compositions comprising the anti-FAP antibodies can be used in therapeutic methods.

The anti-FAP antibodies provided herein are useful for treating diseases characterized by FAP expression, particularly abnormal expression of FAP (e.g., overexpression or a different pattern of expression in a cell) as compared to normal tissue of the same cell type. FAP is aberrantly expressed (e.g., overexpressed) in many human tumors compared to non-tumor tissue of the same cell type. As such, the anti-FAP antibodies provided herein are particularly useful for preventing tumor formation, eradicating tumors, and inhibiting tumor growth or metastasis. The anti-FAP antibodies provided herein can be used to treat any FAP-expressing tumor. Specific malignancies that can be treated by the anti-FAP antibodies provided herein include, for example, lung cancer, colon cancer, stomach cancer, breast cancer, head and neck cancer, skin cancer, liver cancer, kidney cancer, prostate cancer, pancreatic cancer, brain cancer, skeletal muscle cancer.

The anti-FAP antibodies disclosed herein can be used to inhibit tumor growth or kill tumor cells. For example, an anti-FAP antibody can bind to FAP on the cell membrane or cell surface of a cancerous cell (tumor cell or tumor stromal cell) and trigger, for example, ADCC or other effectors to mediate killing of the cancerous cell.

Alternatively, an anti-FAP antibody may be used to block the function of FAP, particularly by physically interfering with its binding to other compounds. For example, the antigen-binding molecules may be used to block FAP enzymatic activity (e.g., serine peptidase, gelatinase, collagenase activity), FAP-mediated degradation of the ECM, and/or FAP-mediated cell invasion or migration.

In one aspect, an anti-FAP antibody for use as a medicament is provided. In still further aspects, anti-FAP antibodies for use in treating diseases characterized by expression of FAP are provided. In certain embodiments, anti-FAP antibodies for use in methods of treatment are provided. In certain embodiments, the invention provides an anti-FAP antibody for use in a method of treating an individual having a disease characterized by expression of FAP, the treatment comprising administering to the individual an effective amount of the anti-FAP antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In still other embodiments, the invention provides anti-FAP antibodies for use in inducing cell lysis. In certain embodiments, the invention provides an anti-FAP antibody for use in a method of inducing cell lysis in an individual, the method comprising administering to the individual an effective amount of the anti-FAP antibody to induce cell lysis. An "individual" according to any of the above embodiments is preferably a human. An "individual" according to any of the above embodiments is preferably a human. The "disease characterized by expression of FAP" according to any of the above embodiments is preferably a cancer, most preferably a cancer selected from the group consisting of: lung cancer, colon cancer, stomach cancer, breast cancer, head and neck cancer, skin cancer, liver cancer, kidney cancer, prostate cancer, pancreatic cancer, brain cancer, skeletal muscle cancer. The "cell" according to any of the above embodiments is preferably a cell present in a tumor, such as a tumor cell or tumor stromal cell, most preferably a tumor cell. "FAP expression" according to any of the above embodiments is preferably abnormal expression, such as overexpression or a different pattern of expression, in a cell compared to normal tissue of the same cell type.

In a further aspect, the invention provides the use of an anti-FAP antibody in the manufacture or manufacture of a medicament. In one embodiment, the medicament is for treating a disease characterized by expression of FAP. In yet another embodiment, the medicament is for use in a method of treating a disease characterized by FAP expression, comprising administering to an individual having a disease characterized by FAP expression an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In yet another embodiment, the medicament is for inducing cell lysis. In yet another embodiment, the medicament is for use in a method of inducing cell lysis in an individual, the method comprising administering to the individual an effective amount of the medicament to induce cell lysis. An "individual" according to any of the above embodiments is preferably a human. The "disease characterized by expression of FAP" according to any of the above embodiments is preferably a cancer, most preferably a cancer selected from the group consisting of: lung cancer, colon cancer, stomach cancer, breast cancer, head and neck cancer, skin cancer, liver cancer, kidney cancer, prostate cancer, pancreatic cancer, brain cancer, skeletal muscle cancer. The "cell" according to any of the above embodiments is preferably a cell present in a tumor, such as a tumor cell or tumor stromal cell, most preferably a tumor cell. "FAP expression" according to any of the above embodiments is preferably abnormal expression, such as overexpression or a different pattern of expression, in a cell compared to normal tissue of the same cell type.

In yet another aspect, the invention provides methods of treating diseases characterized by expression of FAP. In one embodiment, the method comprises administering to an individual having such a disease characterized by expression of FAP an effective amount of an anti-FAP antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. In yet another embodiment, the present invention provides a method for inducing cell lysis in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-FAP antibody to induce cell lysis. An "individual" according to any of the above embodiments may be a human. The "disease characterized by expression of FAP" according to any of the above embodiments is preferably a cancer, most preferably a cancer selected from the group consisting of: lung cancer, colon cancer, stomach cancer, breast cancer, skin cancer, liver cancer, kidney cancer, prostate cancer, pancreatic cancer, brain cancer, skeletal muscle cancer. The "cell" according to any of the above embodiments is preferably a cell present in a tumor, such as a tumor cell or tumor stromal cell, most preferably a tumor cell. "FAP expression" according to any of the above embodiments is preferably abnormal expression, such as overexpression or a different pattern of expression, in a cell compared to normal tissue of the same cell type.

In yet another aspect, the invention provides a pharmaceutical formulation comprising any of the anti-FAP antibodies provided herein, e.g., a pharmaceutical formulation for use in any of the above-described therapeutic methods. In one embodiment, the pharmaceutical formulation comprises any of the anti-FAP antibodies provided herein and one or more pharmaceutically acceptable carriers. In another embodiment, the pharmaceutical formulation comprises any of the anti-FAP antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

The antibodies of the invention may be used alone or in combination with other agents in therapy. For example, an antibody of the invention can be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an anti-cancer agent, such as a chemotherapeutic agent, an inhibitor of tumor cell proliferation, or an activator of tumor cell apoptosis.

Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are contained in the same or different formulations), and separate administration, in which case administration of the antibody of the invention can occur prior to, concurrently with, and/or after administration of the additional therapeutic agent and/or adjuvant. The antibodies of the invention may also be used in combination with radiotherapy.

The antibodies (and any other therapeutic agent) of the invention may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Intravenous administration is generally preferred. However, it is contemplated that the intraperitoneal route is particularly useful, for example, in the treatment of colorectal tumors. Dosing may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is transient or chronic. Various dosing schedules are contemplated herein, including but not limited to a single administration or multiple administrations over multiple time points, bolus administration, and pulse infusion.

The antibodies of the invention should be formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular condition being treated, the particular mammal being treated, the clinical status of the individual patient, the cause, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to practitioners. The antibody need not be, but may optionally be, formulated with one or more drugs currently used to prevent or treat the condition. The effective amount of such other drugs will depend on the amount of antibody present in the formulation, the type of condition being treated, and other factors discussed above. These agents are generally used at the same dosage and with the administration routes described herein, or at about 1-99% of the dosages described herein, or at any dosage and by any route, as empirically determined/clinically determined appropriate.

For the prevention or treatment of disease, the appropriate dosage of the antibody of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, the prophylactic or therapeutic purpose for which the antibody is administered, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitable for administration to a patient in one or a series of treatments. Depending on the type and severity of the disease, about 1. mu.g/kg to 15mg/kg (e.g., 0.1mg/kg to 10 mg/kg) of the antibody may be administered to the patient as a first candidate amount, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dose may range from about 1. mu.g/kg to 100mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment should generally be continued until the desired suppression of the condition occurs. The antibody will range from about 0.05mg/kg to about 10mg/kg at one exemplary dose. Thus, one or more doses of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg or 10mg/kg (or any combination thereof) may be administered to the patient. The above-described dose can be administered intermittently, such as once per week or once every three weeks (e.g., such that the patient receives from about 2 to about 20, or, for example, about 6 doses of antibody). An initial higher loading dose may be administered followed by one or more lower doses. However, other dosing regimens may be used. The progress of the treatment is readily monitored by conventional techniques and assays.

It is to be understood that any of the above formulations or therapeutic methods may be practiced using an antibody conjugate of the invention in place of, or in addition to, an anti-FAP antibody.

H. Article of manufacture

In another aspect of the invention, an article of manufacture is provided that contains materials useful for the treatment, prevention and/or diagnosis of the conditions described above. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials such as glass or plastic. The container contains a composition effective, alone or in combination with another composition, in the treatment, prevention, and/or diagnosis of a condition, and may have a sterile access port (e.g., the container may be a vial or intravenous solution bag having a stopper penetrable by a hypodermic needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates the use of the composition to treat the selected condition. In addition, the article of manufacture can comprise (a) a first container having a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container having a composition contained therein, wherein the composition comprises an additional cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

It will be appreciated that any of the above-described articles of manufacture may comprise an antibody conjugate of the invention in place of or in addition to an anti-FAP antibody.

Example III

The following are examples of the methods and compositions of the present invention. It is to be understood that various other embodiments may be implemented in view of the general description provided above.

Example 1

Recombinant DNA technology

Standard methods are used to manipulate DNA, as described in Sambrook, J.et al, molecular cloning, Alabororymanual, ColdSpringHarbor laboratory Press, ColdSpringHarbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions. The DNA sequence was determined by double-strand sequencing. In some cases, the desired gene segments were prepared from synthetic oligonucleotides and PCR products by automated gene synthesis from GeneartAG (Regensburg, Germany). The gene segment flanked by single restriction endonuclease cleavage sites was cloned into the pGA18(ampR) plasmid. Plasmid DNA was purified from transformed bacteria and the concentration was determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. Gene segments are designed with appropriate restriction sites to allow subcloning into the corresponding expression vector.

General information on the nucleotide sequences of the light and heavy chains of human immunoglobulins is given in Kabat, e.a. et al, (1991) sequencesof proteins of immunologicalcalemtest, fifthed, nihpublications no 91-3242. For expression, all constructs contain a 5' DNA sequence encoding a leader peptide that targets the protein for secretion in eukaryotic cells. Exemplary leader peptides and polynucleotide sequences encoding them are shown in SEQ ID NOs 323 to 331.

(glycoengineered) antibody preparation

The complete antibody heavy and light chain DNA sequences have been obtained by subcloning the variable regions in frame with the constant heavy chain or constant light chain pre-inserted into the corresponding recipient mammalian expression vector. Antibody expression is driven by the MPSV promoter and the vector carries a synthetic polyA signal sequence at the 3' end of the CDS. In addition, each vector contains an EBVORIP sequence.

Antibodies were generated by co-transfecting HEK293-EBNA cells with mammalian antibody expression vectors using calcium phosphate transfection. Exponentially growing HEK293-EBNA cells were transfected by the calcium phosphate method. Alternatively, suspension grown HEK293 cells were transfected with polyethylenimine. To generate unmodified, non-glycoengineered antibodies, cells were transfected with only antibody heavy and light chain expression vectors at a 1:1 ratio.

To generate glycoengineered antibodies, cells were co-transfected with two additional plasmids, one for fusion GnTIII polypeptide expression (GnT-III expression vector) and one for mannosidase II expression (golgi mannosidase II expression vector), at a ratio of 4:4:1:1, respectively. Cells were cultured in T flasks as adherent monolayer cultures using DMEM medium supplemented with 10% FCS and transfected when they were between 50 and 80% confluent. For T150 flask transfection, 25ml DMEM medium supplemented with FCS (final 10% V/V) was inoculated 24 hours prior to transfection at 15X10⁶Isolating the cells, and subjecting the cells to 5% CO₂The atmosphere was left overnight at 37 ℃ in an incubator. For each T150 flask to be transfected, by mixing 94. mu.g total plasmid vector DNA (divided equally between light and heavy chain expression vectors), water (to a final volume of 469. mu.l) and 469. mu.l 1MCaCl₂Preparation of DNA, CaCl from the solution₂And a solution of water. To this solution 938. mu.l 50mM HEPES, 280mM NaCl, 1.5mM Na pH7.05 were added₂HPO₄The solution was immediately mixed for 10 seconds and allowed to stand at room temperature for 20 seconds. The suspension was diluted with 10ml of DMEM supplemented with 2% FCS and added to T150 to replace the existing medium. Then an additional 13ml of transfection medium was added. Cells were incubated at 37 ℃ in 5% CO₂Incubate for about 17 to 20 hours, then replace the medium with 25ml DMEM, 10% FCS. The conditioned medium was harvested approximately 7 days after medium change by centrifugation at 210Xg for 15 min, the solution was sterile filtered (0.22 um filter), sodium azide was added at a final concentration of 0.01% w/v and stored at 4 ℃.

Secreted wild-type or glycoengineered fucosylated antibodies were purified from cell culture supernatants by affinity chromatography using protein a (hitappprota, GEHealthcare) affinity chromatography. Briefly, the cell supernatant was loaded with a 20mM sodium phosphate, 20mM sodium citrate pH7.5 equilibration column, followed by a first wash of 20mM sodium phosphate, 20mM sodium citrate pH7.5 and a second wash of 13.3mM sodium phosphate, 20mM sodium citrate, 500mM sodium chloride pH 5.45. The antibody was eluted with 20mM sodium citrate, 100mM sodium chloride, 100mM glycine pH 3. In a subsequent size exclusion chromatography step on a HiLoadSuperdex200 column (GEHealthcare), the buffer was exchanged for 25mM potassium phosphate, 125mM sodium chloride, 100mM glycine solution pH6.7 or 140mM sodium chloride, 20mM histidine pH6.0 and pure monomeric IgG1 antibody was collected. An additional cation exchange chromatography step is included between the two standard purification steps, if desired.

The protein concentration of the purified protein samples was determined by measuring the Optical Density (OD) at 280nm using the molar extinction coefficient calculated based on the amino acid sequence. SDS-PAGE and Coomassie staining in the presence and absence of reducing agent (5 mM1, 4-dithiothreitol) (SimpleBlue)^TMSafeStain, Invitrogen) to analyze antibody purity and molecular weight. Used according to the manufacturer's instructionsPre-gel systems (Invitrogen, USA) (4-20% Tris-glycine gels or 3-12% Bis-Tris). Analytical size exclusion column (GEHealthcare, Sweden) using Superdex20010/300GL at 2mM OPS, 150mM NaCl, 0.02% NaN₃The aggregate content of the antibody samples was analyzed at 25 ℃ in running buffer at ph 7.3. After elimination of N-glycans by peptide-N glycosidase f (roche molecular biochemicals) enzyme treatment, the integrity of the amino acid backbone of the reduced antibody light and heavy chains was verified by nanoelectrospray q-TOF mass spectrometry.

The results of purification and analysis of wild-type and glycoengineered 28H1, 29B11, 3F2, and 4G8 human IgG antibodies are shown in fig. 15 to 22. The yields are given in the following table:

oligosaccharides attached to the Fc region of the antibody were analyzed by maldtof-MS, as described below. Oligosaccharides were enzymatically released from the antibodies by PNGaseF digestion. The resulting digest solution containing the released oligosaccharides is either prepared directly for MALDITOF-MS analysis or is further digested with EndoH glycosidase followed by sample preparation for MALDITOF-MS analysis.

Sugar Structure analysis of (glycoengineered) antibodies

To determine the relative proportion of oligosaccharide structures containing fucose and non-fucose (a-fucose), the glycans released by the purified antibody material were analyzed by MALDI-Tof mass spectrometry. Antibody samples (approximately 50. mu.g) were incubated with 5 mUN-glycosidase F (QABio; PNGaseF: E-PNG01) overnight at 37 ℃ in 2mM Tris pH7.0 to release oligosaccharides from the protein backbone. To determine glycans, acetic acid was added to a final concentration of 150mM and incubated at 37 ℃ for 1 hour. For analysis by MALDITOF mass spectrometry, 2 μ L of the sample was mixed on a MALDI target with 2 μ LDHB matrix solution (2, 5-dihydroxybenzoic acid [ Bruker Daltonics #201346] dissolved at 4mg/ml in 50% ethanol/5 mM NaCl) and analyzed with a MALDITOF mass spectrometer Autoflex II instrument [ Bruker Daltonics ]. Routinely, 50-300 shots are recorded and summed up as one experiment. The spectra obtained were evaluated by flexanalysis software (bruker daltonics) and the mass determined for each peak detected. Subsequently, peaks are assigned to carbohydrate structures containing fucose or a-fucose (non-fucose) by calculating masses and theoretically expected masses by comparing the respective structures (e.g. complex, hybrid and oligo or high mannose, with and without fucose, respectively).

To determine the proportion of hybrid structures, N-glycosidase F and endoglycosidase H [ QAbio; EndoH E-EH02 samples of antibodies were treated. N-glycosidase F releases all N-linked glycan structures (complex, hybrid, and oligo-and high mannose structures) from the protein backbone, while endoglycosidase H additionally cleaves all hybrid-type glycans between two N-acetylglucosamine (GlcNAc) residues at the glycan-reducing terminus. This digest was then processed in the same manner as described above for the N-glycosidase F digested sample and analyzed by maldtof mass spectrometry. The degree of reduction in signal for a particular carbohydrate structure was used to estimate the relative content of hybrid structures by comparing the patterns from the N-glycosidase F digest and the combined N-glycosidase F/endoh digest. The relative amount of each carbohydrate structure was calculated from the ratio of the peak height of each structure to the sum of the peak heights of all oligosaccharides tested. The amount of fucose refers to the percentage of fucose-containing structures relative to all carbohydrate structures (e.g., complexes, hybrids, and oligo and high mannose structures, respectively) identified in the N-glycosidase F treated sample. The amount of nonfucosylation refers to the percentage of fucose-deficient structures relative to all carbohydrate structures identified in the N-glycosidase F-treated sample (e.g., complexes, hybrids, and oligo and high mannose structures, respectively).

The degree of nonfucosylation of different wild-type and glycoengineered anti-FAP antibodies is shown in the following table:

example 2

Construction of Universal Fab libraries

A Fab-format universal antibody library was constructed on the basis of human germline genes using the following V-domain pairings: vk3_20 kappa light chain and VH3_23 heavy chain for the DP47-3 library and Vk1_17 kappa light chain and VH1_69 heavy chain for the DP88-3 library. See seq id nos1 and 2.

Both libraries were randomized in the light chain CDR3(L3) and the heavy chain CDR3(H3), and each library was assembled from 3 fragments by overlapping extended Splicing (SOE) PCR. Fragment 1 contains the 5 'end of the antibody gene, including randomized L3, fragment 2 is a central constant fragment, spanning L3 to H3, and fragment 3 contains randomized H3 and the 3' portion of the antibody gene.

The following primer combinations were used to generate library fragments of the DP47-3 library: fragment 1(LMB 3-LibL 1b _ new), fragment 2(MS 63-MS 64), fragment 3(Lib 2H-fdseqlong). See table 3. The following primer combinations were used to generate library fragments of the DP88-3 library: segment 1(LMB 3-RJH _ LIB3), segment 2(RJH 31-RJH 32) and segment 3(LIB88_ 2-fdsolong). See table 4.

TABLE 3

TABLE 4

The PCR protocol used to generate the library fragments included: initial denaturation at 94 ℃ for 5 min; 1 minute 94 ℃,1 minute 58 ℃, and 1 minute 72 ℃ for 25 cycles; and 10 minutes final extension at 72 ℃. For assembly PCR, 3 fragments in equimolar ratio were used as templates. The assembly PCR protocol included: initial denaturation at 94 ℃ for 3 min; and 5 cycles of 94 ℃ for 30 seconds, 58 ℃ for 1 minute, and 72 ℃ for 2 minutes. At this stage, primers complementary to sequences outside of fragments 1-3 were added and 20 more cycles were performed, followed by 10 minutes of final extension at 72 ℃.

After a sufficient amount of full-length randomized Fab construct was assembled, the Fab construct was digested with NcoI/NotI (for the DP47-3 library) and with NcoI/NheI (for the DP88-3 library) while the recipient phagemid vector was similarly treated. For the DP47-3 library, 22.8. mu.g of the Fab library was ligated with 16.2. mu.g of phagemid vector. For the DP88-3 library, 30.6. mu.g of the Fab library was ligated with 30.6. mu.g of the phagemid vector.

The purified ligation system was used for 68 DP47-3 library transformations and 64 DP88-3 library transformations, respectively, to obtain a final library capacity of DP47-3 of 4.2X10¹⁰And DP88-3 Final library Capacity 3.3X10⁹. Phagemid particles displaying the Fab library were rescued and purified by PEG/NaCl purification for selection.

Example 3

Selection of anti-FAP clones (Primary selection)

Selection was made for the extracellular domain of human or murine Fibroblast Activation Protein (FAP) cloned upstream of polylysine and the 6xhis tag. See SEQ ID NOs 317 and 319. Before selection, antigens were coated into the immune tubes at a concentration of 10. mu.g/ml or 5. mu.g/ml depending on the selection round. The following protocol was followed for selection: (i) about 10¹²Individual library DP47-3 phagemid particles bound to immobilized human or murine FAP for 2 hours; (ii) washing the immune tubes with 5x5ml PBS/Tween20 and 5x5ml PBS; (iii) the phage particles were eluted by adding 1mL of 100mM TEA (triethylamine) for 10 minutes and neutralized by adding 500. mu.L of 1M Tris/HClpH7.4; and (iv) reinfection of log phase Escherichia coli TG1 cells, infection with helper phage VCSM13, followed by PEG/NaCl precipitation of phagemid particles for subsequent selection rounds.

More than 3 or4 rounds of selection were performed using human FAP at decreasing antigen concentrations and in some cases murine FAP was used at 5 μ g/ml in the last round of selection. Specific binders were defined as signals 5-fold higher than background and were identified by ELISA. NUNCmaxisorp plates were coated with 10 μ g/ml human or murine FAP, followed by addition of Fab-containing bacterial supernatant and detection of specifically bound fabs via their Flag tag using an anti-Flag/HRP secondary antibody.

ELISA positive clones were expressed by bacteria as 1mL cultures in a 96-well plate format and the supernatants were subjected to kinetic screening experiments using biacore 100. Measurement of K by surface plasmon resonance_DI.e. useT100 machine (GEHealthcare) with anti-human F (ab') immobilized on CM5 chips by amine coupling at 25 ℃₂The fragment specifically captures the antibody (Jackson ImmunoResearch # 109-. Briefly, carboxymethylated dextran biosensor chips (CM5, GEHealthcare) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Anti-human F (ab')₂Fragment-specific capture antibodiesThe conjugate capture protein was diluted to 50. mu.g/ml with 10mM sodium acetate, pH5, and then injected at a flow rate of 10. mu.l/min to achieve approximately up to 10.000 Response Units (RU). After injection of the capture antibody, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, Fab or purified Fab from bacterial supernatants was injected at 10 μ Ι/min for 300 seconds and dissociated for 300 seconds to achieve capture baseline stabilization. The capture level was in the range of 100-500 RU. In a subsequent step, a human or murine FAP analyte diluted in HBS-EP + (ge healthcare,10mm phes, 150mm naci, 3mm edta,0.05% surfactant p20, ph7.4) either in single concentration or in a range of concentrations (depending on the clonal affinity in the range between 100nM and 250 pM) was injected at 25 ℃ at a flow rate of 50 μ l/min. The binding time was 120 or 180 seconds and the dissociation time was 300 to 600 seconds. The surface of the sensor was regenerated by injecting glycine at 90. mu.l/min pH1.5 for 30 seconds followed by NaOH at the same flow rate for 20 seconds. Using a simple one-to-one langmuir binding model (T100evaluation software or Scrubber software (BioLogic)) the association rate (k) was calculated by simultaneous fitting of association and dissociation sensorgrams_on) And dissociation rate (k)_off). At a ratio of k_off/k_onCalculation of equilibrium dissociation constant (K)_D)。

Example 4

Construction of an anti-FAP affinity maturation library

Three affinity maturation libraries were constructed based on pre-selected antibodies from the primary anti-FAP selection. More precisely, they are based on (i) anti-human FAP clone 2D9 (library a.m. FAP2d 9) (see seq id nos. 229 and 231), (ii) anti-murine FAP clone 4B8 (library a.m. FAP4B 8) (see seq id nos. 233 and 235) and (iii) cross-reactive clones 7a1, 13B2, 13C2, 13E8, 14C10 and 17a11 (library a.m. fappool) (see seq id nos. 237 and 239, variable region sequences corresponding to 7a1, seq id nos. 241 and 243, variable region sequences corresponding to 13C2, seq id nos. 245 and 247, variable region sequences corresponding to 13E 8; seq id nos. 249 and 251, variable region sequences corresponding to 14C10, and seq id nos. 253 and 255, variable region sequences corresponding to 17a 11).

Each of these libraries consisted of two sub-libraries, randomized in the light chain CDR1 and CDR2 (L1/L2) or in the heavy chain CDR1 and CDR2 (H1/H2), respectively. These sub-libraries are merged at the time of transformation. Each of these sub-libraries was constructed by four subsequent steps of amplification and assembly.

For the L1/L2 library, the amplification and assembly protocol included: (i) fragment 1(LMB 3-DPK 22_ CDR1_ rand _ ba _ opt) and fragment 2 (DPK 22_ CDR1_ fo-DPK 22_ Ck _ BsiWI _ ba) were amplified; (ii) assembling fragments 1 and 2 using the outer primers LMB3 and DPK22_ Ck _ BsiWI _ ba to create a template for fragment 3; (iii) fragment 3 (LMB 3-DPK 22_ CDR2_ rand _ ba) and fragment 4 (DPK 22_ CDR2_ fo-DPK 22_ Ck _ BsiWI _ ba) were amplified; and (iv) final assembly of fragments 3 and 4 using the same outer primers as above. The primer sequences are shown in Table 5.

TABLE 5

Bold: 60% initial base and 40% randomization to M

Underlining: 60% initial base and 40% randomization to N

For the H1/H2 library, the amplification and assembly protocol included: (i) amplifying segment 1 (RJH 53-DP 47_ CDR1_ rand _ ba _ opt) and segment 2 (DP 47_ CDR1_ fo-MS 52); (ii) assembling fragments 1 and 2 using the outer primers RJH53 and MS52 to create a template for fragment 3; (iii) amplifying segment 3 (RJH 53-DP 47_ CDR2_ rand _ ba) and segment 4 (DP 47_ CDR2_ fo-MS 52); and (iv) final assembly of fragments 3 and 4 using the same outer primers as above. The primer sequences are shown in Table 6.

TABLE 6

Bold: 60% initial base and 40% randomization to M

Underlining: 60% initial base and 40% randomization to N

The final assembled product was digested with NcoI/BsiWI (L1/L2 sub-library for a.m.fap2d9 and a.m.fap4b 8) and MunI/NheI (H1/H2 sub-library for a.m.fap2d9 and a.m.fap4b 8) respectively, as well as with NcoI/BamHI (L1/L2 library for a.m.fappool) and BspEI/PstI (H1/H2 library for a.m.fappool) together with similarly processed acceptor vectors based on plasmid preparations of equimolar mixtures of clones 2D9, 4B8 or clones 7a1, 13B2, 13C2, 13E8, 14C10 and 17a11 respectively. The following amounts of digested randomized (partial) V-domain and digested receptor vector (μ gV domain/μ g vector) were ligated for each library: a.m.FAP2D9L1/L2 sub-library (5.7/21.5), a.m.FAP2D9H1/H2 sub-library (4.1/15.5), a.m.FAP4B8L1/L2 sub-library (6.5/24.5), a.m.FAP4B8H1/H2 sub-library (5.7/21.5), a.m.FAP poolL1/L2 sub-library (4.4/20), and a.m.FAP poolH1/H2 sub-library (3.4/15.5).

For each of the 3 affinity maturation libraries, the purified L1/L2 and H1/H2 sublibrary ligation were combined and used for 60 transformations to obtain a final library capacity of 6.2X10⁹(for a.m. fap2d 9), 9.9x10⁹(for a.m.FAP4 B8) and 2.2x10⁹(for a.m. fappool).

Construction of additional affinity maturation libraries against FAP (based on clones 3F2, 3D9, 4G8, 4B3 and 2C 6)

Cross-reactive antibodies pre-selected at the first affinity maturation stage from anti-FAP antibodies (i.e.clones 3F2, 3D9, 4G8, 4B3 and 2C 6; see SEQ ID NOs: 195 and 197, variable region sequence corresponding to 3F 2; SEQ ID NOs: 199 and 199; and 2C 6)201, variable region sequence corresponding to 3D 9; 205 and 207, corresponding to the variable region sequence of 4G 8; 209 and 211, corresponding to the variable region sequence of 4B 3; 217 and 219, corresponding to the variable region sequence of 2C 6) four additional affinity maturation libraries were constructed. More precisely, these four libraries were based on 1) anti-FAP clones 3F2, 4G8 and 4B3 (V)_HLibrary, randomized in CDRs 1 and 2 of variable heavy chain, i.e., H1/H2 library), 2) anti-FAP clones 3D9 and 2C6 (V)_LThe library was randomized in CDRs 1 and 2 of the variable light chain, i.e., the L1/L2 library), 3) anti-FAP clone 3F2 (the L3 library, soft randomization in CDR3 of the light chain, i.e., the L3 library) and 4) anti-FAP clone 3F2 (the H3 library, soft randomization in CDR3 of the heavy chain, i.e., the H3 library). The first two libraries were constructed in exactly the same way as the methods performed on the L1/L2 and H1/H2 libraries, respectively, at the first affinity maturation of the anti-FAP antibody. In comparison, for the L3 and H3 affinity maturation libraries based on clone 3F2, two new primers were used at L3 of the parental clone (AM-3F 2-DPK 22-L3-ba: CACTTTGGTCCCCTGGCCGAACGT)CGGGGGAAGCATAATACCCTGCTGACAGTAATACACTGC, where the underlined bases are 60% given base and 40% mix N (mixture of four nucleotides A, C, G, and T)) and H3 (AM _3F2_ DP47_ H3_ fo: GGCCGTATATTACTGTGCGAAAGGGTGGTTTGGTGGTTTTAACTACTGGGGCCAAGGAAC, where the underlined bases are 60% given and 40% mixture N, the italicized bases are 60% given and 40% G, and the underlined italicized bases are 60% given and 40% mixture K (mixture of two nucleotides G and T)). The library capacity is as follows: H1/H2 library (1.13X 10)¹⁰) L1/L2 library (5.6X 10)⁹) L3 library (2.3X 10)¹⁰) And H3 library (2.64X 10)¹⁰）。

Example 5

Selection of affinity matured anti-FAP clones

Human or murine fibroblast activation protein directed against the 5' clone of polylysine and the 6xhis tag(FAP) to select. See SEQ ID NOs 317 and 319. Before selection, the antigen was coated into the immune tubes at a concentration of 10. mu.g/ml, 5. mu.g/ml or 0.2. mu.g/ml depending on the library and round of selection. The following protocol was followed for selection: (i) about 10¹²Individual library FAP2D9, a.m. FAP4b8 or a.m. fappool phagemid particles bind to immobilized human or murine FAP for 2 hours; (ii) wash the immune tubes with 10-20x5ml pbs/Tween20 and 10-20x5ml pbs (based on the library and selection round); (iii) the phage particles were eluted by adding 1mL of 100mM TEA (triethylamine) for 10 minutes and neutralized by adding 500. mu.L of 1M Tris/HClpH7.4; and (iv) reinfection of log phase Escherichia coli TG1 cells, infection with helper phage VCSM13, followed by PEG/NaCl precipitation of phagemid particles for subsequent selection rounds.

More than 2 rounds of selection were performed and conditions were individually adjusted for each of the 3 libraries. In detail, the selection parameters are: m.fap2d9 (round 1,5 μ g/mL of human FAP and a total of 20 washes, round 2,1 μ g/mL of human FAP and a total of 30 washes), a.m.fap4b8 (round 1,1 μ g/mL of murine FAP and a total of 30 washes, round 2, 0.2 μ g/mL of human FAP and a total of 40 washes) and a.m.fappool (round 1,5 μ g/mL of human FAP and a total of 30 washes, round 2,5 μ g/mL of murine FAP and a total of 30 washes). Specific binders were defined as signals 5-fold higher than background and were identified by ELISA. NUNCmaxisorp plates were coated with 1 μ g/ml or 0.2 μ g/ml human or murine FAP, followed by addition of Fab-containing bacterial supernatant and detection of specifically bound fabs via their Flag tag using an anti-Flag/HRP secondary antibody.

ELISA positive clones were expressed by bacteria as 1mL cultures in a 96-well plate format and then subjected to kinetic screening experiments on the supernatants using biacore 100 as described above (see example 3).

Selection of additional affinity matured anti-FAP clones

Selection was made for human and murine Fibroblast Activator Protein (FAP) extracellular domains cloned upstream of 6x lysine and 6x-his tags (see SEQ ID NOs: 317 and 319). Before selection, antigens were coated at any concentration of 1. mu.g/ml, 0.2. mu.g/ml or 0.02. mu.g/ml according to the library and selection roundIn epidemic control. Selection and ELISA-based screening were performed as described for the first affinity maturation stage of the anti-FAP antibody. Secondary screening was performed using a ProteOnXPR36 biosensor (Biorad) and kinetic rate constants and affinities were determined by analyzing affinity purified Fab preparations on the same instrument. K was measured by surface plasmon resonance using a ProteOnXPR36 instrument (Biorad) at 25 ℃ with anti-human F (ab') 2 fragment specific capture antibodies immobilized on a GLM chip (Jackson ImmunoResearch #109-005-006) and subsequent capture of Fab from bacterial supernatants or from purified Fab preparations_D. Briefly, the GLM biosensor chip (Biorad) was activated for 5 minutes with a freshly prepared mixture of N-ethyl-N' - (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Anti-human F (ab') 2 fragment-specific capture antibodies were diluted to 24. mu.g/ml with 10mM sodium acetate pH5.0 followed by injection for 5 minutes to achieve approximately up to 10.000 Response Units (RU) of conjugated capture antibody. After injection of the capture antibody, 1M ethanolamine was injected for 5 minutes to block unreacted groups. For kinetic measurements, Fab from bacterial supernatant was injected at a flow rate of 30. mu.l/min for 100 seconds. The capture level was in the range of 250 RU. In a subsequent capture, serial dilutions (two-fold dilution, highest concentration 25 nM) of human, murine or cynomolgus FAP analyte diluted in PBS/0.005% Tween-20 were injected at 25 ℃ at a flow rate of 50 μ l/min. The binding time was 240 seconds and the dissociation time was 600 to 1800 seconds. By injecting 0.85% H at 100. mu.l/min₃PO₄30 seconds followed by injection of 50mM NaOH30 seconds at the same flow rate to regenerate the chip. The binding rate (k) was calculated by simultaneous fitting of the binding and dissociation sensorgrams using a simple one-to-one Langmuir binding model (ProteOn management software version 2.1)_on) And dissociation rate (k)_off). At a ratio of k_off/k_onTo balance the dissociation constant (K)_D)。

The following affinity matured clones were identified: 19G1 (see seq id no:257 and 259), 20G8 (see seq id no:281 and 263), 4B9 (see seq id no:265 and 267), 5B8 (see seq id no:269 and 271), 5F1 (see seq id no:273 and 275), 14B3 (see seq id no:277 and 279), 16F1 (see seq id no:281 and 283), 16F8 (see seq id no:285 and 287), O3C9 (see seq id no:289 and 291), 22a3 (see seq id no:301 and 303) and 29B11 (see seq id no:305 and 307) (all of these clones were selected from the H42/H2 library and were derived from the parent clone 3F 2), O2D7 (see seq id no:293 and 295) (based on the L3 and 1 of the parent antibody), 20G8 (see seq id no:281 and 2) and all of these clones were selected from the parent clone 4624 and 309 (see seq id no: 4624).

Figures 1 to 5 show surface plasmon resonance sensorgrams of selected affinity matured Fab binding to immobilized FAP, while table 7 gives the corresponding affinities deduced. The selected fabs span the high affinity range (pM to nM range) and are cross-reactive to human (hu) and murine (mu) FAP and cynomolgus (cyno) FAP as determined for the selected clones. Affinity matured anti-fafab was converted to Fab-IL2-Fab format and IgG antibodies for specificity analysis. Binding specificity was shown by not binding to DPPIV expressed on HEK293 or CHO cells, which is an intimate homologue of FAP (see example 9).

TABLE 7

Summary of kinetic equilibrium constants (KD) (monovalent binding) of affinity matured anti-FAP antibodies in Fab fragment form

Example 6

IgG conversion of Fab binding to FAP

Parent 3F2, 4G8 and 3D9Fab and affinity matured 3F2 and 4G8Fab derivatives were converted into the human IgG1, mouse IgG2a and human IgG1 formats.

The complete antibody heavy and light chain DNA sequences are obtained by subcloning the variable regions in frame with the corresponding heavy and light chain constant regions previously inserted into different recipient mammalian expression vectors, or by recombination by fusing short sequence strings homologous to the recipient vector insertion sites. Recombination was performed according to the "In-fusion cloning System" from Invitrogen.

Antibody expression was driven by the MPSV promoter in all vectors, and all vectors carried a synthetic polyA signal sequence at the CDS 3' end. In addition, each vector contains an EBVOriP sequence.

Example 7

Biacore analysis of anti-fapgg antibodies

Then used at 25 ℃The T100 instrument (GEHealthcare) determined and confirmed the affinity of anti-fapp fab fragments 3F2, 4G8 and 3D9 and human IgG1 for transforming anti-FAP antibodies to human, murine and cynomolgus FAP by Surface Plasmon Resonance (SPR) analysis. For this purpose, the human, mouse or cynomolgus FAP ectodomain (seq id nos 317-322) was captured by an immobilized anti-His antibody (PentaHisQiagen34660) and the antibody was used as analyte. For immobilization, carboxymethylated dextran biosensor chips (CM5, GEHealthcare) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The PentaHis antibody was diluted to 40. mu.g/ml with 10mM sodium acetate pH5, followed by injection at a flow rate of 10. mu.l/min to achieve approximately 9000 Response Units (RU) of the conjugated protein. After injection of ligand, 1M ethanolamine was injected to block unreacted groups.

For kinetic measurements, human, mouse or cynomolgus FAP extracellular domain was injected at 10 μ l/min, 10nM for 20 seconds (for Fab fragments) or 20nM for 25 seconds (for IgG), and captured via its His-tag by an immobilized 5xHis antibody. A serial dilution of antibody (two-fold serial dilution of Fab fragment) was injected at 25 ℃ at a flow rate of 90. mu.l/min in HBS-EP + (GEHealthcare,10mM HEPES,150mM NaCl,3mM EDTA,0.05% surfactant P20, pH7.4)In the range of 6.25nM to 200nM, or IgG in 5-fold serial dilutions in the range of 3.2pM to 10 nM). The following parameters were applied: binding time 180 seconds, dissociation time 300 seconds (for Fab) or 900 seconds (for IgG), between each cycle 60 seconds 10mM glycine pH2 regeneration. Use of a simple one-to-one Langmuir binding model by simultaneous fitting of binding and dissociation sensorgrams ((S))T100evaluation software version 1.1.1) calculation of the binding Rate (k)_on) And dissociation rate (k)_off) (model parameters are local Rmax and RI = 0). At a ratio of k_off/k_onTo calculate equilibrium dissociation maturation (K)_D)。

Table 8 gives bound K_DThe value is obtained. FIGS. 6A-C show the corresponding SPR-based kinetic analysis of Fab fragments, and FIGS. 7A-C show the kinetic analysis of IgG antibodies.

TABLE 8

Summary of kinetic equilibrium constants (KD) of Fab fragments and IgG forms of 3F2, 4G8, and 3D9 anti-FAP antibodies

Construction article	Human FAP	Mouse FAP	Kiwi FAP
				IgG3F2	Affinity: 39pM	Affinity: 29pM	Affinity: 42pM
IgG4G8	Affinity: 51pM	Affinity: 1pM	Affinity: 59pM
				IgG3D9	Affinity: 93pM	Affinity: 96pM	Affinity: 96pM
Fab fragment 3F2	Affinity: 13nM	Affinity: 14nM	Affinity: 11nM
				Fab fragment 4G8	Affinity: 74nM	Affinity: 7nM or less	Affinity: 56nM
Fab fragment 3D9	Affinity: 133nM	Affinity: 32nM	Affinity: 143nM

Example 8

Binding of anti-FAP antibodies 3F2, 4G8 and 3D9 on human tumor tissue sections

We performed experiments to detect and compare FAP expression in freshly frozen human tumor tissues (breast cancer, colon adenocarcinoma and NSCLC tissue) using anti-FAP antibody clones 3F2, 4G8 and 3D9 (as mouse IgG2 a).

A fresh frozen Tissue Microarray (TMA) (AST274) from the rochel (r) clinical & tissue biomarkers tumor bank, containing 30 different tumors, two spots each, was used. TMA containing 10 invasive breast ductal carcinomas, 10 colorectal adenocarcinomas and 10 non-small cell lung carcinomas were obtained from AsterandLtd, Royston, UK.

For Immunohistochemical (IHC) staining, the following antibodies were used: monoclonal mouse anti-human FAP clone 3F2 (15.8 ng/ml, diluted in Ventana antibody diluent), monoclonal mouse anti-human FAP clone 4G8 (1000 ng/ml, diluted in Ventana antibody diluent), and monoclonal mouse anti-human FAP clone 3D9 (1000 ng/ml, diluted in Ventana antibody diluent). Polyclonal mouse IgG2a (concentration 100. mu.g/mL) (supplier: DAKO, X0943, batch No. 00058066) was used as isotype control.

Staining was performed according to standard protocols on a ventana benhmarkxt instrument using the ventana ultra-View detection kit and HRP detection system (containing universal hrpmultimer, and DAB for staining). Counterstaining was performed with hematoxylin II (Ventana, Mayer's hematoxylin) and Blueing reagent (Ventana) for 8 minutes.

TMA was semi-quantitatively analyzed and tumor tissue was assessed for total FAP expression (staining intensity) and localization of FAP expression.

With all three anti-FAP antibodies, all tumor tissue samples (breast, colorectal and lung cancers) that could be evaluated showed moderate to strong staining for FAP signal intensity in the stromal component of the tumor. At least 7 out of 10 samples per tumor and antibodies can be evaluated. The remaining samples could not be evaluated because the tissue core had folding artifacts (foldingartifacts), contained only normal tissue, or was missing.

As expected, the FAP signal is invariably localized in the stromal component of the tumor. There was a slight difference in signal intensity between clone 3F2 and clones 3D9 and 4G 8. Slightly stronger signals were seen with clones 3D9 and 4G8, however the difference was minor.

Figures 8A-D show representative photomicrographs of human tumor tissue samples immunohistochemically stained for FAP using anti-FAP mouse IgG2a3F2, 3D9, or 4G8, or isotype control antibodies.

Example 9

Binding of anti-FAP antibodies to FAP on cells

Binding of human IgG1 antibodies 3F2, 4B3, and 4G8 to human and murine FAP expressed on stably transfected HEK293 cells was measured by FACS. Briefly, 150.000 cells per well were incubated with the anti-FAP antibodies 3F2, 4B3, and 4G8 at the indicated concentrations for 30 minutes at 4 ℃ in round bottom 96-well plates and washed once with PBS/0.1% BSA. Bound antibodies were detected using FACSCANTIII (software FACSDiva) with FITC-conjugated Affinipure goat anti-human F (ab ') 2 specific F (ab') 2 fragment (Jackson Immunoresearch Lab #109-096-097, working solution: 1:20 dilution in PBS/0.1% BSA, freshly prepared) incubated for 30 min at 4 ℃.

The results are shown in FIG. 9. EC50 values at half maximal binding to human and murine FAP were determined and are given in table 9.

TABLE 9

Binding of anti-Fab antibodies to FAP on cells (EC 50 values)

Specificity of FAP antibodies

To assess the binding specificity of the phage display derived antibodies, the anti-FAP human IgG1 antibodies 3F2, 4B3 and 4G8 measured binding to HEK293 cells stably expressing DPPIV (an intimate homolog of FAP expressed on healthy tissue) or HER 2. Briefly, 200.000 cells per well (HEK 293-DPPIV or HEK293-HER2 as controls) were incubated with 30 μ G/ml anti-FAP antibodies 3F2, 4B3 or 4G8 in round bottom 96 well plates for 30 minutes at 4 ℃ and washed once with PBS/0.1% BSA. Trastuzumab (anti-HER 2 antibody) or Phycoerythrin (PE) conjugated mouse anti-human anti-CD 26/DPPIV antibody (CD26 = DPPIV, mouse IgG1, k, BDBiosciences, #555437, clone M-a 261) was used as a positive control. Bound antibodies were detected using FACSCANTIII (software FACSDiva) with PE-conjugated Affinipure goat anti-human IgGFc gamma specific F (ab') 2 fragment (Jackson Immunoresearch Lab #109-116-170, working solution: 1:20 dilution in PBS/0.1% BSA, freshly prepared) incubated for 30 min at 4 ℃. The results of this experiment are shown in figure 10. None of the anti-FAP antibodies showed significant binding to DPPIV or HER2, but the signal was in the range of the negative control (secondary antibody only, isotype control antibody, or no antibody at all).

Binding of anti-FAP antibodies to FAP on human fibroblasts

Binding of human IgG1 antibody to human FAP expressed on human fibroblast cell line GM05389 (derived from human fetal lung, national institute of general medical sciences, Camden, NJ) was measured by FACS. Briefly, 200.000 cells per well were incubated with 30 μ G/ml anti-FAP antibody 3F2 or 4G8 in round bottom 96 well plates for 30 minutes at 4 ℃ and washed once with PBS/0.1% BSA. Bound antibodies were detected using FACSCANTIII (software FACSDiva) with FITC-conjugated Affinipure goat anti-human IgGFc gamma specific F (ab') 2 fragment (Jackson Immunoresearch Lab #109-096-098, working solution: 1:20 dilution in PBS/0.1% BSA, freshly prepared) incubated for 30 min at 4 ℃. The results of this experiment are shown in figure 11. Both anti-FAP antibodies strongly bind to FAP expressed on human fibroblasts.

Binding of anti-FAP antibodies to FAP on human tumor cells

The binding of human IgG1 antibody to human FAP expressed on human fibroblast cell line GM05389 and on stably transfected HEK293 cells was compared to FAP expression on human cancer cell lines ACHN, Colo205, MDA-MB231, MDA-MB435, and KPL4 by FACS.

Briefly, 200.000 cells per well were incubated with 10 μ G/ml anti-FAP antibody 3F2 or 4G8 in round bottom 96 well plates for 30 minutes at 4 ℃ and washed once with PBS/0.1% BSA. Bound antibodies were detected using FACSCANTIII (software FACSDiva) with FITC-conjugated Affinipure goat anti-human F (ab ') 2 specific F (ab') 2 fragment (Jackson Immunoresearch Lab #109-096-097, working solution: 1:20 dilution in PBS/0.1% BSA, freshly prepared) incubated for 30 min at 4 ℃. The results of this experiment are shown in figure 12. The data show that antibodies 3F2 and 4G8 specifically bind to FAP that is strongly overexpressed on fibroblasts and stably transfected HEK293 cells; whereas only weak binding was detected on ACHN, Colo205, MDA-MB231, MDA-MB435 and KPL4 human tumor cell lines.

Example 10

Analysis of FAP internalization by FACS for anti-FAP antibody binding

For several antibodies to FAP known in the art, they have been described as inducing FAP internalization upon binding (described, e.g., in baumet, jdrugtarget15,399-406(2007); baueretal, journal of clinical oncology,2010 ascon meetingproceedings (Post-MeetingEdition), vol.28(May20Supplement), astractno.13062 (2010); ostrermanentanal, clinocerr Res14,4584-4592 (2008)).

Thus, we analyzed the internalization properties of our antibodies. Briefly, GM05389 cells (human lung fibroblasts) cultured in EMEM medium +15% FCS were dissociated, washed, counted, checked for viability, and seeded at a density of 0.2mio cells/well in 12-well plates. The following day, FAP antibodies 4G8 and 3F2 (fig. 13A) or only 4G8 (fig. 13B) were diluted to 10 μ G/ml in cold medium, the cells were allowed to cool on ice, and diluted antibodies (0.5 ml/well) or only medium were added as indicated. Subsequently, the cells were incubated in a cold chamber for 30 minutes at moderate temperature and shaking, followed by the addition of 0.5ml of warm medium and further incubation of the cells at 37 ℃ for the indicated period of time. When different time points were reached, the cells were transferred to ice, washed once with cold PBS, and incubated with 0.4ml secondary antibody (AlexaFluor633 conjugated goat anti-human IgG, molecular probes # A-21091,2mg/ml,1:500 for 30 min) at 4 ℃. Then, cells were washed twice with PBS/0.1% BSA, transferred to 96-well plates, centrifuged at 400xg for 4 minutes at 4 ℃, and cell pellet resuspended by vortex shaking. Cells were fixed using 100 μ l of 2% PFA. For FACS measurements, cells were resuspended in 200. mu.l/sample PBS/0.1% BSA and measured in a plate protocol in FACSCAntoII (software FACSDiva). The results of these experiments are presented in fig. 13A and B, and show that 4G8 and 3F2 anti-FAP antibodies do not induce FAP internalization on fibroblasts.

Analysis of FAP internalization by immunofluorescence upon anti-FAP antibody binding

GM05389 cells (human lung fibroblasts) were cultured on glass coverslips in EMEM medium +15% FCS. Prior to treatment, cells were washed three times with PBS and starved for 2 hours in EMEM medium +0.1% BSA. anti-FAP antibody (4G 8 IgG) or anti-CD 20 antibody (GA 101, used as isotype control) was diluted to a final concentration of 10 μ G/ml in cold EMEM medium. After starvation, cells were cooled on ice, rinsed twice with cold PBS, and incubated with diluted antibodies (0.5 ml/well) at 4 ℃ for 45 minutes under constant shaking to allow surface binding. The cells were then washed twice with cold PBS and either fixed with cold PFA (T0, 4% paraformaldehyde in pbsph7.4) or further incubated in EMEM +10% FCS at 37 ℃ for 20 min, 1 h, 3 h and 6 h. At each time point, cells were washed twice with cold PBS and PFA fixed on ice for 20 min. After fixation, cells were washed four times with cold PBS, permeabilized with triton0.03%, and incubated with anti-EEA 1 (early endosomal marker) antibody in blocking buffer (PBS +10% FCS) for 45 min at room temperature. Then, cells were washed three times with PBS and further incubated with fluorescently labeled secondary antibodies (donkey anti-mouse AlexaFluor594 conjugated antibody, and goat anti-human AlexaFluor488 conjugated antibody) for 45 minutes at room temperature. Finally, the cells were washed and mounted on glass-supported slides using ImmunoMount mounting medium.

Fig. 14A-D present representative immunofluorescence images showing FAP plasma membrane staining on GM05389 lung fibroblasts obtained after 4 ℃ 45 min (a), 37 ℃ 20 min (B), 37 ℃ 1 hr (C), or 37 ℃ 6 hr (D) anti-FAP 4G8IgG binding. The anti-CD 20 antibody GA101 used as isotype control showed background staining. EEA1 marks the early endosome. Note that FAP surface plasma membrane staining lasts up to 6 hours after binding of anti-FAP 4G8 antibody.

Example 11

Biacore analysis of affinity matured anti-FAPAIgG antibodies

Affinity matured anti-fafab fragments derived from 3F2 and 4G8 were converted into rabbit IgG antibodies. Affinity of affinity matured rabbit IgG1 based on 3F2 and 4G8, was subsequently determined and confirmed by SPR analysis (Biacore) at 25 ℃ to convert anti-FAP antibodies to human, murine and cynomolgus FAP. For this purpose, the human, mouse or cynomolgus FAP ectodomain (seq id nos 317-322) was captured by an immobilized anti-His antibody (PentaHisQiagen34660) and the antibody was used as analyte. IgG1:5 was diluted from 10nM to 3.2 pM. The following parameters were applied: the binding time was 180 seconds, the dissociation time was 900 seconds, and the flow was 90. mu.l/min. Regeneration was carried out with 10mM glycine pH2 for 60 seconds. Fitting the curve with a 1:1 model to obtain K_DValue (R max local, RI = 0).

Example 12

Binding of affinity matured anti-FAP antibodies to FAP on cells

The binding of the affinity mature human IgG1 antibody 28H1 (1.89 mg/ml) to human FAP expressed on stably transfected HEK293 cells derived from the Alexa-647 marker (1.83 moles dye per mole protein) of the 4G8 parent antibody was measured by FACS. Briefly, 200.000 cells per well were incubated with the indicated concentrations of 2. mu.g/ml and 10. mu.g/ml parental 4G8 and affinity matured 28H1 anti-FAP antibody in round bottom 96 well plates for 30 minutes at 4 ℃ and washed once with PBS/0.1% BSA. Bound antibodies were detected using facscan ii (software facsdiva) incubated for 30 minutes at 4 ℃. The data show that both antibodies strongly bind to HEK293 cells transfected with human FAP (fig. 23).

Example 13

Binding of affinity matured anti-FAP antibodies to FAP on human fibroblasts

Binding of affinity matured human IgG1 antibody derived from 3F2 to human FAP expressed on the human fibroblast cell line GM05389 (derived from human fetal lung, national institute of general medical sciences, Camden, NJ) was measured by FACS. Briefly, 200.000 cells per well were incubated with 30 μ g/ml affinity matured 3F2 anti-FAP antibody in round bottom 96 well plates for 30 minutes at 4 ℃ and washed once with PBS/0.1% BSA. Bound antibodies were detected using FACSCANTIII (software FACSDiva) with FITC-conjugated Affinipure goat anti-human IgGFc gamma specific F (ab') 2 fragment (Jackson Immunoresearch Lab #109-096-098, working solution: 1:20 dilution in PBS/0.1% BSA, freshly prepared) incubated for 30 min at 4 ℃. EC50 values were determined for binding to human and murine FAP at half maximal binding.

Example 14

Antibody-dependent cell-mediated cytotoxicity mediated by glycoengineered anti-fapgg 1 antibodies

Human IgG1 antibodies against FAP derived from 4G8 or 3F2 were glycoengineered by co-transfection of plasmids encoding GnTIII and ManII as described in example 1. Subsequently, glycoengineered parent 4G8 and 3F2 and affinity matured 28H1 human IgG1 antibodies were compared in an ADCC assay for their potential to mediate superior antibody-mediated cellular cytotoxicity compared to their non-glycoengineered wild-type form. Briefly, HEK293 cells stably transfected with human FAP as target cells were collected, washed and resuspended in culture medium, stained with freshly prepared calcein am (molecular probes) for 30 min at 37 ℃, washed three times, counted, and diluted to 300.000 cells/ml. This suspension was transferred to a round bottom 96-well plate (30.000 cells/well), the corresponding antibody dilution was added, and incubated for 10 minutes to push the test antibody to bind the cells before contacting with effector cells. For PBMC, the ratio of effector cells to target cells is 25 to 1. The co-incubation was performed for 4 hours. As readout, Lactate Dehydrogenase (LDH) released into the supernatant after disruption of the challenged cells was determined. LDH was collected from the co-culture supernatant and analyzed with an LDH detection kit (roche applied science). The substrate conversion of LDH enzyme was measured with an ELISA absorbance reader (SoftMaxPro software, reference wavelength: 490nm vs. 650 nm). As shown in figure 24, all anti-FAP antibodies tested were able to induce ADCC against HEK 293-hfp cells. The performance of the glycoengineered (ge) form was consistently superior to the corresponding wild-type (wt) non-glycoengineered form.

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Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims (36)

1. An antibody that specifically binds to Fibroblast Activation Protein (FAP), wherein the antibody comprises

(i) A heavy chain variable region comprising heavy chain CDR1 of SEQ ID NO. 3, heavy chain CDR2 of SEQ ID NO. 47, and heavy chain CDR3 of SEQ ID NO. 135 and a light chain variable region comprising light chain CDR1 of SEQ ID NO. 143, light chain CDR2 of SEQ ID NO. 151, and light chain CDR3 of SEQ ID NO. 163;

(ii) a heavy chain variable region comprising heavy chain CDR1 of SEQ ID NO.13, heavy chain CDR2 of SEQ ID NO. 81, and heavy chain CDR3 of SEQ ID NO. 135 and a light chain variable region comprising light chain CDR1 of SEQ ID NO. 143, light chain CDR2 of SEQ ID NO. 151, and light chain CDR3 of SEQ ID NO. 163;

(iii) a heavy chain variable region comprising heavy chain CDR1 of SEQ ID NO. 23, heavy chain CDR2 of SEQ ID NO. 113, and heavy chain CDR3 of SEQ ID NO. 135, and a light chain variable region comprising light chain CDR1 of SEQ ID NO. 143, light chain CDR2 of SEQ ID NO. 151, and light chain CDR3 of SEQ ID NO. 163;

(iv) a heavy chain variable region comprising heavy chain CDR1 of SEQ ID NO. 9, heavy chain CDR2 of SEQ ID NO. 61, and heavy chain CDR3 of SEQ ID NO. 137 and a light chain variable region comprising light chain CDR1 of SEQ ID NO. 147, light chain CDR2 of SEQ ID NO. 155, and light chain CDR3 of SEQ ID NO. 167;

(v) a heavy chain variable region comprising heavy chain CDR1 of SEQ ID NO. 19, heavy chain CDR2 of SEQ ID NO. 95, and heavy chain CDR3 of SEQ ID NO. 137 and a light chain variable region comprising light chain CDR1 of SEQ ID NO. 147, light chain CDR2 of SEQ ID NO. 155, and light chain CDR3 of SEQ ID NO. 167; or

(vi) A heavy chain variable region comprising heavy chain CDR1 of SEQ ID NO. 31, heavy chain CDR2 of SEQ ID NO. 127, and heavy chain CDR3 of SEQ ID NO. 137 and a light chain variable region comprising light chain CDR1 of SEQ ID NO. 147, light chain CDR2 of SEQ ID NO. 155, and light chain CDR3 of SEQ ID NO. 167.

2. The antibody of claim 1, wherein the antibody of items (i) - (iii) comprises the heavy chain variable region of SEQ ID NO:267 and the light chain variable region of SEQ ID NO: 265.

3. The antibody of claim 1, wherein the antibody of items (iv) - (vi) comprises the heavy chain variable region of SEQ ID NO:299 and the light chain variable region of SEQ ID NO: 297.

4. The antibody of any one of claims 1 to 3, wherein the antibody comprises an Fc region of an immunoglobulin.

5. An antibody that specifically binds to Fibroblast Activation Protein (FAP), wherein the antibody comprises

(i) A heavy chain variable region comprising heavy chain CDR1 of SEQ ID NO. 3, heavy chain CDR2 of SEQ ID NO. 35, and heavy chain CDR3 of SEQ ID NO. 137 and a light chain variable region comprising light chain CDR1 of SEQ ID NO. 147, light chain CDR2 of SEQ ID NO. 155, and light chain CDR3 of SEQ ID NO. 167;

(ii) a heavy chain variable region comprising heavy chain CDR1 of SEQ ID NO.13, heavy chain CDR2 of SEQ ID NO. 69, and heavy chain CDR3 of SEQ ID NO. 137 and a light chain variable region comprising light chain CDR1 of SEQ ID NO. 147, light chain CDR2 of SEQ ID NO. 155, and light chain CDR3 of SEQ ID NO. 167;

(iii) a heavy chain variable region comprising heavy chain CDR1 of SEQ ID NO. 23, heavy chain CDR2 of SEQ ID NO. 101, and heavy chain CDR3 of SEQ ID NO. 137 and a light chain variable region comprising light chain CDR1 of SEQ ID NO. 147, light chain CDR2 of SEQ ID NO. 155, and light chain CDR3 of SEQ ID NO. 167;

(iv) a heavy chain variable region comprising heavy chain CDR1 of SEQ ID NO. 3, heavy chain CDR2 of SEQ ID NO. 35, and heavy chain CDR3 of SEQ ID NO. 135, and a light chain variable region comprising light chain CDR1 of SEQ ID NO. 143, light chain CDR2 of SEQ ID NO. 151, and light chain CDR3 of SEQ ID NO. 163;

(v) a heavy chain variable region comprising heavy chain CDR1 of SEQ ID NO.13, heavy chain CDR2 of SEQ ID NO. 69, and heavy chain CDR3 of SEQ ID NO. 135 and a light chain variable region comprising light chain CDR1 of SEQ ID NO. 143, light chain CDR2 of SEQ ID NO. 151, and light chain CDR3 of SEQ ID NO. 163; or

(vi) A heavy chain variable region comprising heavy chain CDR1 of SEQ ID NO. 23, heavy chain CDR2 of SEQ ID NO. 101, and heavy chain CDR3 of SEQ ID NO. 135, and a light chain variable region comprising light chain CDR1 of SEQ ID NO. 143, light chain CDR2 of SEQ ID NO. 151, and light chain CDR3 of SEQ ID NO. 163,

and the antibody comprises an Fc region of an immunoglobulin.

6. The antibody of claim 5, wherein the antibody of items (i) - (iii) comprises the heavy chain variable region of SEQ ID NO 207 and the light chain variable region of SEQ ID NO 205.

7. The antibody of claim 5, wherein the antibody of items (iv) - (vi) comprises the heavy chain variable region of SEQ ID NO:197 and the light chain variable region of SEQ ID NO: 195.

8. The antibody of any one of claims 4-7, wherein the Fc region is an IgGFc region.

9. The antibody of any one of claims 1 to 8, wherein the antibody is a full length IgG class antibody.

10. The antibody of any one of claims 1 to 9, wherein the antibody comprises a human constant region.

11. The antibody of any one of claims 1 to 10, wherein the antibody is a human antibody.

12. The antibody of any one of claims 1 to 11, wherein the antibody comprises a glycoengineered Fc region.

13. The antibody of claim 12, wherein the antibody has an increased proportion of nonfucosylated oligosaccharides in the Fc region as compared to a nonglycoengineered antibody.

14. The antibody of claim 12 or 13, wherein at least 20% to 100% of the N-linked oligosaccharides in the Fc region are nonfucosylated.

15. The antibody of any one of claims 12 to 14, wherein the antibody has an increased proportion of bisected oligosaccharides in the Fc region as compared to a non-glycoengineered antibody.

16. The antibody of any one of claims 12 to 15, wherein at least 20% to 100% of the N-linked oligosaccharides in the Fc region are bisected.

17. The antibody of any one of claims 12 to 16, wherein at least 20% to 50% of the N-linked oligosaccharides in the Fc region are bisected, nonfucosylated.

18. The antibody of any one of claims 1 to 17, wherein the antibody has increased effector function and/or increased Fc receptor binding affinity.

19. The antibody of claim 18, wherein the increased effector function is increased ADCC.

20. One or more isolated polynucleotides encoding the antibody heavy chain and the antibody light chain of an antibody according to any one of claims 1 to 19.

21. An isolated polypeptide encoded by the polynucleotide of claim 20.

22. A composition comprising (i) a first isolated polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:267 and a second isolated polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:265, (ii) a first isolated polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:299 and a second isolated polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:297, (iii) a first isolated polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:197 and a second isolated polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:195, or (iv) a first isolated polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:207 and a second isolated polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:207, the second isolated polynucleotide encodes a polypeptide comprising the sequence of seq id No. 205.

23. A vector comprising the polynucleotide of claim 20.

24. A host cell comprising the polynucleotide of claim 20, the composition of claim 22, or the vector of claim 23.

25. The host cell of claim 24, wherein said host cell is manipulated to express elevated levels of one or more polypeptides having GnTIII activity.

26. The host cell of claim 25, wherein said polypeptide having GnTIII activity is a fusion polypeptide comprising the catalytic domain of GnTIII and the Golgi localization domain of ManII.

27. The host cell of claim 25 or 26, wherein said host cell is further manipulated to express elevated levels of one or more polypeptides having manll activity.

28. A method of generating an antibody that specifically binds to Fibroblast Activation Protein (FAP), the method comprising

a) Culturing the host cell of claim 24 in a culture medium under conditions permitting expression of the antibody, and

b) recovering the antibody.

29. A method of generating an antibody that specifically binds to Fibroblast Activation Protein (FAP), the method comprising

a) Culturing the host cell of any one of claims 25 to 27 in a culture medium under conditions that allow expression of the antibody and modification of oligosaccharides present on the Fc region of said antibody by said polypeptide having GnTIII activity, and

b) recovering the antibody.

30. An antibody that specifically binds FAP, wherein the antibody is generated by the method of claim 28 or 29.

31. An antibody conjugate comprising the antibody of any one of claims 1 to 19 and a cytotoxic agent.

32. A pharmaceutical composition comprising the antibody of any one of claims 1 to 19 and a pharmaceutically acceptable carrier.

33. The pharmaceutical composition of claim 32, further comprising an additional therapeutic agent.

34. Use of an antibody according to any one of claims 4 to 19 in the manufacture of a medicament for the treatment of breast, colon, kidney or lung cancer.

35. The use of claim 34, wherein the treatment further comprises administering an additional therapeutic agent.

36. Use of an antibody according to any one of claims 1 to 19 in the preparation of a diagnostic agent for diagnosing breast, colon, kidney or lung cancer in an individual, wherein the diagnostic agent comprises an antibody according to any one of claims 1 to 19 and a label that allows the detection of a complex of the diagnostic agent and FAP.