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  Pharmaceutical Patents  

 

Title:  Anti-CD26 antibodies and methods of use thereof
United States Patent: 
8,030,469
Issued: 
October 4, 2011

Inventors: 
Aoyagi; Teikichi (Saitama, JP), Luo; Peter Peizhi (Lansdale, PA), Zhong; Pingyu (Blue Bell, PA), Hsieh; Mark (Jenkintown, PA), Li; Yan (San Jose, CA), Wang; Kevin Caili (Lansdale, PA), Morimoto; Chikao (Setagaya-ku, JP)
Assignee: 
SBI Incubation Co., Ltd. (Tokyo, JP)
Appl. No.:  12/263,919
Filed:
 November 3, 2008


 

Training Courses -- Pharm/Biotech/etc.


Abstract

The present invention provides novel anti-CD26 antibodies and other, related polypeptides, as well as novel polynucleotides encoding the antibodies and polypeptides. The invention also provides methods of making the antibodies and polypeptides. Compositions and cells comprising the antibodies or polypeptides are further provided. Methods of using the antibodies and/or polypeptides, such as to inhibit cell proliferation and in the treatment of conditions associated with CD26, are also provided.

Description of the Invention

The present invention provides a variety of novel polypeptides including those comprising one or more CDRs or FRs of an anti-CD26 antibody or comprising a heavy chain variable region or light chain variable region (or fragment thereof) of an anti-CD26 antibody. In some embodiments, the polypeptides bind CD26. In particular, a variety of novel anti-CD26 antibodies are provided, including, but not limited to, humanized anti-CD26 antibodies. Compositions, such as pharmaceutical compositions comprising the polypeptides are also provided. Polynucleotides encoding the polypeptides and vectors and host cells comprising the polynucleotides are also provided. Methods of making and using the polypeptides are also provided. In some instances, the polypeptides of the invention are useful as intermediates for making, for example, polypeptides (such as antibodies) that bind CD26.

It is understood that wherever embodiments are described herein with the language "comprising," otherwise analogous embodiments described in terms of "consisting of" and/or "consisting essentially of" are also provided.

General Techniques

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995).

Polypeptides

The invention provides a variety of novel polypeptides comprising one or more heavy chain and/or light chain complementarity determining regions (CDRs), heavy chain and/or light chain framework regions (FRs), and/or heavy chain and/or light chain variable regions (VHs and VLs, respectively). In some embodiments, the polypeptides are antibodies. In some embodiments, the polypeptides are isolated. The invention also encompasses polypeptides which are substantially pure. In some embodiments, the antibodies are monoclonal antibodies. In some embodiments, the antibodies are chimeric antibodies. In some embodiments, the antibodies are humanized antibodies. In some embodiments, the antibodies are human antibodies. In some embodiments, the polypeptides are not (i.e., are other than) murine monoclonal antibodies. In some embodiments, the polypeptides are not (i.e., are other than) mouse monoclonal antibodies. In some embodiments, the polypeptides are not (i.e., are other than) the 14D10 antibody (Dong et al. (1998) Mol. Immunol. 35(1):13-21 and U.S. Pat. Pub. No. 2003/0031665). In some embodiments, the polypeptides do not comprise the VH or the VL (or both the VH and VL) or a particular CDR, FR, set of CDRs, or set of FRs of the 14D10 antibody. In some embodiments, the polypeptides do not comprise one or more of the CDRs and/or one or more of the FRs of the 14D10 antibody. In some embodiments, the polypeptides do not comprise all of the CDRs and/or all of the FRs of the 14D10 antibody. In some embodiments, the polypeptides do not comprise both the VH and VL of the 14D10 antibody. (The VH and VL and the CDRs and FRs of the 14D10 antibody are identified in FIGS. 3 and 5 (see Original Patent).) In some embodiments, the polypeptide does not comprise a heavy chain variable region of the sequence QVKLQESGPGLVQPSQTLSITCTVSGFSLTTYGVHWVRQSPGKGLEWLGVIWGGGRTD YDAAFISRLSISKDNSKSQVFFKMNSLQANDTAIYYCVRNRHDWFDYWGQGTTVTVSS (CM03 VH; SEQ ID NO:90). In some embodiments, the polypeptide does not comprise a light chain variable region of the sequence DIQMTQSPSSLSASLGDRVTITCSASQGIRNSLNWYQQKPDGAVKLLIYYSSNLHSGVPS RFSGSGSGTDFSLTISNLEPEDIATYYCQQSIKLPFTFGSGTKLEIK (CM03 VL; SEQ ID NO:91). In some embodiments, the polypeptide does not comprise both a heavy chain variable region of the sequence QVKLQESGPGLVQPSQTLSITCTVSGFSLTTYGVHWVRQSPGKGLEWLGVIWGGGRTD YDAAFISRLSISKDNSKSQVFFKMNSLQANDTAIYYCVRNRHDWFDYWGQGTTVTVSS (SEQ ID NO:90) and a light chain variable region of the sequence DIQMTQSPSSLSASLGDRVTITCSASQGIRNSLNWYQQKPDGAVKLLIYYSSNLHSGVPS RFSGSGSGTDFSLTISNLEPEDIATYYCQQSIKLPFTFGSGTKLEIK (SEQ ID NO:91). In some embodiments of each of the aforementioned aspects, as well as other aspects described herein, the polypeptide is not the mouse monoclonal antibody 1F7 (U.S. Pat. No. 5,120,642; PCT Publication No. WO 91/07985 (Schlossman et al.); Morimoto et al. (1989) J. Immunology, 143:3430-3439). In some embodiments, the polypeptides do not comprise both the VH and VL of the 1F7 antibody. In some embodiments, the polypeptides do not comprise one or more of the CDRs and/or one or more of the FRs of the 1F7 antibody. In some embodiments, the polypeptides do not comprise all of the CDRs and/or all of the FRs of the 1F7 antibody. In some embodiments, the polypeptides are affinity matured antibodies. In some embodiments, a polypeptide described herein is an antibody chain, such as a heavy chain or light chain. In some embodiments, the polypeptide comprises a light chain variable region (e.g., a light chain variable region comprising one or more of the light chain CDRs described herein and/or one or more of the light chain FRs described herein). In some embodiments, the polypeptide comprises a heavy chain variable region (e.g., a heavy chain variable region comprising one or more of the heavy chain complementarity determining regions (CDRs) described herein and/or one or more of the light chain framework regions (FRs) described herein). In some embodiments, the invention encompasses polypeptides which each comprise both a light chain variable region and a heavy chain variable region. The invention further encompasses polypeptides which are intermediates in the synthesis of antibodies or other CD26-binding polypeptides.

In some embodiments, the polypeptides, such as antibodies, described herein bind CD26. In some embodiments, the polypeptides described herein preferentially bind CD26. In some embodiments, the polypeptides bind human CD26. In some embodiments, the polypeptides preferentially bind human CD26. In some embodiments, the polypeptides crossreact with human CD26 and CD26 of another species. In some embodiments, a polypeptide described herein binds human CD26 with a K.sub.D of about 200 nM or less, about 60 nM or less, about 30 nM or less, about 12 nM or less, about 6 nM or less, or about 3 nM, or about 1 nM or less. In some embodiments, the polypeptide binds human CD26 with a K.sub.D of about 10 nM or less. In some embodiments, the polypeptide binds human CD26 with a K.sub.D of about 6 nM or less. In some embodiments, the polypeptide binds human CD26 with a K.sub.D of about 3 nM or less. In some embodiments, the polypeptide binds human CD26 with a K.sub.D of about 1 nM or less. In some embodiments, the polypeptide (e.g., antibody) binds human CD26 with a K.sub.D of about 0.1 nM to about 10 nM, about 0.1 nM to about 6 nM, about 0.1 nM to about 3 nM, or about 0.1 nM to about 1 nM.

In some embodiments, the polypeptides (e.g., antibodies) described herein bind to one or more peptides selected from the group consisting of YSLRWISDHEYLY (SEQ ID NO:45; peptide 6), LEYNYVKQWRHSY (SEQ ID NO:46; peptide 35), TWSPVGHKLAYVW (SEQ ID NO:47; peptide 55), LWWSPNGTFLAYA (SEQ ID NO:48; peptide 84), RISLQWLRRIQNY (SEQ ID NO:49; peptide 132), YVKQWRHSYTASY (SEQ ID NO:50; peptide 37), EEEVFSAYSALWW (SEQ ID NO:51; peptide 79), DYSISPDGQFILL (SEQ ID NO:52; peptide 29), SISPDGQFILLEY (SEQ ID NO:53; peptide 30), and IYVKIEPNLPSYR (SEQ ID NO:54; peptide 63). In some embodiments, the polypeptides described herein preferentially bind to the one or more peptides. These peptides are regions of human CD26. In some embodiments, the polypeptides described herein bind to the same epitope as the mouse monoclonal antibody 14D10. In some embodiments, the polypeptides described herein are capable of blocking the binding of mouse monoclonal antibody 14D10 to human CD26 in a competition assay. In some embodiments, the polypeptides described herein are capable of blocking the binding of mouse monoclonal antibody 1F7 to human CD26 in a competition assay.

Methods of determining affinity are known in the art. For instance, binding affinity may be determined using a BIAcore biosensor, a KinExA biosensor, scintillation proximity assays, ELISA, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence transfer, and/or yeast display. Binding affinity may also be screened using a suitable bioassay.

One way of determining binding affinity of antibodies to CD26 is by measuring affinity of monofunctional Fab fragments of the antibodies. To obtain monofunctional Fab fragments, antibodies, for example, IgGs can be cleaved with papain or expressed recombinantly. Affinities of anti-CD26 Fab fragments of monoclonal antibodies can be determined by Surface Plasmon Resonance (SPR) system (BIAcore 3000.TM., BIAcore, Inc., Piscaway, N.J.). SA chips (streptavidin) are used according to the supplier's instructions. Biotinylated CD26 can be diluted into HBS-EP (100 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% P20) and injected over the chip at a concentration of 0.005 mg/mL. Using variable flow time across the individual chip channels, two ranges of antigen density are achieved: 10-20 response units (RU) for detailed kinetic studies and 500-600 RU for concentration. A mixture of Pierce elution buffer and 4 M NaCl (2:1) effectively removes the bound Fab while keeping the activity of CD26 on the chip for over 200 injections. HBS-EP buffer can be used as running buffer for all the BIAcore assays. Serial dilutions (0.1-10.times. estimated K.sub.D) of purified Fab samples are injected for 2 min at 100 .mu.L/min and dissociation times of up to 30 h min are generally allowed. The concentrations of the Fab proteins can be determined by ELISA and/or SDS-PAGE electrophoresis using a standard Fab of known concentration (determined by amino acid analysis). Kinetic association rates (k.sub.on) and dissociation rates (k.sub.off) are obtained simultaneously by fitting the data to a 1:1 Langmuir binding model (Lofas & Johnsson, 1990) using the BIAevaluation program. Equilibrium dissociation constant (K.sub.D) values are calculated as k.sub.off/k.sub.on.

In some embodiments, the invention encompasses polypeptides, such as antibodies, which inhibit proliferation of cells expressing CD26. The invention also encompasses embodiments where the polypeptides are useful in the treatment of a condition (such as a disease or disorder) associated with CD26 expression (e.g., a T-cell malignancy). In some embodiments, the polypeptides (e.g., antibodies) of the invention may have one or more of the following characteristics: (a) bind CD26; (b) modulate CD26 activity, (c) cause cell cycle arrest of CD26+ cells at the G1/S checkpoint; (d) inhibit proliferation of cells expressing CD26, and/or (e) are useful in the treatment of a condition associated with CD26 expression. In some embodiments, the polypeptides are useful in inhibiting growth of a CD26-expressing tumor, inducing regression of a CD26-expressing tumor, and/or inhibiting metastasis of CD26 expressing cancer cells. In some embodiments, the condition associated with CD26 expression is a disease or disorder associated with CD26 overexpression. In some embodiments, the condition associated with CD26 expression is mediated, at least in part, by CD26. In some embodiments, the condition associated with CD26 expression is a condition associated with the proliferation of cells expressing CD26. In some embodiments, the disease or disorder is a cancer (e.g., a solid tumor cancer, pancreatic cancer, kidney cancer, or lymphoma), an autoimmune disease or disorder, graft versus host disease (GVHD), or an inflammatory disease or disorder.

In one aspect, the invention provides a polypeptide comprising one or more (e.g., one, two, three, four, five or six) complementarity determining regions (CDRs) described herein. In another aspect, the invention provides a polypeptide comprising one or more (e.g., one, two, or three) heavy chain complementarity determining regions (CDRs) described herein and/or one or more (e.g., one, two, or three) light chain CDRs described herein. In some of the embodiments, the polypeptide further comprises one or more (e.g., one, two, three, or four) heavy chain framework regions (FRs) described herein and/or one or more (e.g., one, two, three, or four) light chain FRs described herein. In another aspect, the invention provides a polypeptide comprising a heavy chain variable region comprising one or more (e.g., one, two, or three) heavy chain complementarity determining regions (CDRs) described herein and/or a light chain variable region comprising one or more (e.g., one, two, or three) light chain CDRs described herein. In some of the embodiments, the heavy chain variable region further comprises one or more (e.g., one, two, three, or four) heavy chain framework regions (FRs) described herein and/or the light chain variable region further comprises one or more (e.g., one, two, three, or four) light chain FRs described herein.

It is understood that reference to "heavy chain CDR(s)" or "light chain CDR(s)" does not mean that all embodiments of these CDRs are contained in a heavy chain or light chain, respectively. These terms are used for convenience to indicate their origin. The invention, however, does include embodiments in which one or more CDRs are contained within (a) heavy chain variable region(s) and/or light chain variable region(s), and/or (b) heavy chain(s) and/or light chain(s). The same principles apply to framework designations.

The invention provides a polypeptide, such as an antibody, comprising: (a) one or more (e.g., one, two, or three) heavy chain CDRs that each have at least about 80% identity to a CDR selected from the group consisting of (i) a heavy chain CDR1 selected from the group consisting of GFSLTTYGVH (SEQ ID NO:55), GFSLSTYGVH (SEQ ID NO:56), and GYSLTTYGVH (SEQ ID NO:57), (ii) a heavy chain CDR2 selected from the group consisting of VIWGDGRTDYDAAFMS (SEQ ID NO:58) and VIWGDGRTDYDSSFMS (SEQ ID NO:59), and (iii) a heavy chain CDR3 sequence NRHDWFDY (SEQ ID NO:60); and/or (b) one or more (e.g., one, two, or three) light chain CDRs that each have at least about 80% identity to a CDR selected from the group consisting of (i) a light chain CDR1 selected from the group consisting of RASQDIRNNLN (SEQ ID NO:61), RASQGIRNNLN (SEQ ID NO:62), and SASQDIRNSLN (SEQ ID NO:63), (ii) a light chain CDR2 selected from the group consisting of YSSNLHS (SEQ ID NO:64), YSSNLQS (SEQ ID NO:65) and YSSNLHT (SEQ ID NO:66), and (iii) a light chain CDR3 selected from the group consisting of QQSIKLPLT (SEQ ID NO:67), QQSIKLPFT (SEQ ID NO:68), and QQSNKLPLT (SEQ ID NO:69). In some embodiments, the CDR(s) have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% identity to the indicated sequence(s). In some embodiments, the polypeptide binds CD26. In some embodiments, the polypeptide is not a murine monoclonal antibody. In some embodiments, the polypeptide is an antibody that comprises: (i) a heavy chain CDR1 that has at least about 80% identity to GFSLTTYGVH (SEQ ID NO:55), (ii) a heavy chain CDR2 that has at least about 80% identity to VIWGDGRTDYDAAFMS (SEQ ID NO:58), and (iii) a heavy chain CDR3 that has at least about 80% identity to NRHDWFDY (SEQ ID NO:60). In some embodiments, the polypeptide is an antibody that comprises (i) a light chain CDR1 that has at least about 80% identity to RASQGIRNNLN (SEQ ID NO:62), (ii) a light chain CDR2 that has at least about 80% identity to YSSNLQS (SEQ ID NO:65), and (iii) a light chain CDR3 that has at least about 80% identity to QQSIKLPFT (SEQ ID NO:68).

In some embodiments, the polypeptide comprises one or more (e.g., one, two, or three) CDRs, wherein each of the CDRs comprises a CDR of a heavy chain variable region (VH) X384, X385, X386, X387, X388, X399, or X420 shown in FIG. 5. In some embodiments, the polypeptide comprises one or more (e.g., one, two, or three) CDRs, wherein each of the CDRs comprises a CDR of a light chain variable region (VL) shown in FIG. 3 (see Original Patent) as any of the sequences X376, X377, X378, X379, X380, X381, or X394. In some embodiments, the one or more CDRs are CDR1, CDR2, or CDR3 of the light chain variable region or heavy chain variable region shown in FIG. 3 or 5 (see Original Patent), respectively (e.g., CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and/or CDR-H3). Some embodiments include polypeptides (such as antibodies) that comprise one or more CDRs of a heavy chain variable region (VH) sequence X384, X385, X386, X387, X388, X399, or X420 shown in FIG. 5, as well as one or more CDRs of a light chain variable region sequence X376, X377, X378, X379, X380, X381, or X394 shown in FIG. 3.

In some embodiments, the invention provides a polypeptide, such as an antibody, which comprises at least one CDR that is at least about 80% identical to at least one CDR, at least two CDRs, or at least three CDRs of a VH shown in FIG. 5 as any of the sequences X384, X385, X386, X387, X388, X399, or X420 or of a light chain variable region (VL) shown in FIG. 3 as any of the sequences X376, X377, X378, X379, X380, X381, or X394. Other embodiments include polypeptides which comprise at least two or three CDR(s) that are at least about 80% identical to at least two or three CDRs of a VH shown in FIG. 5 as any of the sequences X384, X385, X386, X387, X388, X399, or X420 or a VL shown in FIG. 3 as any of the sequences X376, X377, X378, X379, X380, X381, or X394. In some embodiments, one or more CDRs substantially homologous to at least one CDR, at least two, or at least three CDRs of a VH or VL shown in FIG. 5 or 3 are at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to at least one, at least two, or at least three CDRs of a VH or VL shown in FIG. 5 as any of the sequences X384, X385, X386, X387, X388, X399, or X420 or shown in FIG. 3 in any of the sequences X376, X377, X378, X379, X380, X381, or X394. In some embodiments, the CDR is a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and/or CDR-H3.

Determination of CDRs is well within the skill of the art. In some embodiments, the CDRs are Kabat CDRs. In other embodiments, the CDRs are Chothia CDRs. In still further embodiments, one or more of the CDRs are defined by a combination of the Kabat and Chothia definitions.

In one aspect, the invention provides a polypeptide comprising: (a) a heavy chain CDR1 comprising the sequence GX.sub.1X.sub.2LX.sub.3TYGVH (SEQ ID NO:31), wherein X.sub.1 is F or Y, X.sub.2 is S or T, and X.sub.3 is T, N, or S; (b) a heavy chain CDR2 comprising the sequence VIWGX.sub.1GRTDYDX.sub.2X.sub.3FMS (SEQ ID NO:32), wherein X.sub.1 is G or D, X.sub.2 is A or S, and X.sub.3 is A or S; and/or (c) a heavy chain CDR3 comprising the sequence X.sub.1RHDWFDY (SEQ ID NO:33), wherein X.sub.1 is N or S. In some embodiments, the polypeptide comprises the CDR1. In some embodiments, the polypeptide comprises the CDR2. In some embodiments, the polypeptide comprises the CDR3. In some embodiments, the polypeptide comprises the CDR1, the CDR2, and the CDR3. In some embodiments, the polypeptide comprises a heavy chain variable region comprising the CDR1, the CDR2, and/or the CDR3. In some embodiments, the polypeptide further comprises a light chain variable region (e.g., a light chain variable region described herein).

In some embodiments, the polypeptide comprises heavy chain CDRs comprising (i) a CDR1 comprising the sequence GX.sub.1X.sub.2LX.sub.3TYGVH (SEQ ID NO:31), wherein X.sub.1 is F or Y, X.sub.2 is S or T, and X.sub.3 is T, N, or S; (ii) a CDR2 comprising the sequence VIWGX.sub.1GRTDYDX.sub.2X.sub.3FMS (SEQ ID NO:32), wherein X.sub.1 is G or D, X.sub.2 is A or S, and X.sub.3 is A or S; and (iii) a CDR3 comprising the sequence X.sub.1RHDWFDY (SEQ ID NO:33), wherein X.sub.1 is N or S. In some embodiments, the polypeptide comprises a heavy chain variable region comprising the heavy chain CDRs.

In some embodiments, the polypeptide comprises a heavy chain CDR1 comprising a sequence selected from the group consisting of GFSLTTYGVH (SEQ ID NO:55), GFSLSTYGVH (SEQ ID NO:56), and GYSLTTYGVH (SEQ ID NO:57). In some embodiments, the polypeptide comprises a heavy chain CDR2 comprising a sequence selected from the group consisting of VIWGDGRTDYDAAFMS (SEQ ID NO:58) and VIWGDGRTDYDSSFMS (SEQ ID NO:59). In some embodiments, the polypeptide comprises a heavy chain CDR3 comprising the sequence NRHDWFDY (SEQ ID NO:60). In some embodiments, the polypeptide comprises a heavy chain variable region comprising the heavy chain CDR(s).

Again, where any aspect or embodiment of the invention is described herein in terms of a Markush group or other grouping of alternatives, the present invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, but also the main group absent one or more of the group members. The present invention also envisages the explicit exclusion of one or more of any of the group members in the claimed invention.

In some embodiments, the polypeptide comprises heavy chain CDRs comprising: (a) a CDR1 comprising the sequence GFSLTTYGVH (SEQ ID NO:55); (b) a CDR2 comprising the sequence VIWGDGRTDYDAAFMS (SEQ ID NO:58); and (c) a CDR3 comprising the sequence NRHDWFDY (SEQ ID NO:60). In some embodiments, the polypeptide comprises heavy chain CDRs comprising: (a) a CDR1 comprising the sequence GFSLSTYGVH (SEQ ID NO:56); (b) a CDR2 comprising the sequence VIWGDGRTDYDAAFMS (SEQ ID NO:58); and (c) a CDR3 comprising the sequence NRHDWFDY (SEQ ID NO:60). In some embodiments, the polypeptide comprises heavy chain CDRs comprising: (a) a CDR1 comprising the sequence GYSLTTYGVH (SEQ ID NO:57); (b) a CDR2 comprising the sequence VIWGDGRTDYDSSFMS (SEQ ID NO:59); and (c) a CDR3 comprising the sequence NRHDWFDY (SEQ ID NO:60). In some embodiments of each of the above, the polypeptide comprises a heavy chain variable region that comprises the specified CDR1, CDR2, and CDR3.

In another aspect, the invention provides a polypeptide comprising: (a) a light chain CDR1 comprising the sequence X.sub.1ASQX.sub.2IRNX.sub.3LN (SEQ ID NO:34), wherein X.sub.1 is S or R, X.sub.2 is G or D, and X.sub.3 is S or N; (b) a light chain CDR2 comprising the sequence YSSNLX.sub.1X.sub.2 (SEQ ID NO:35), wherein X.sub.1 is H or Q and X.sub.2 is S or T; and/or (c) a light chain CDR3 comprising the sequence QQSX.sub.1KLPX.sub.2T (SEQ ID NO:36), wherein X.sub.1 is I or N and X.sub.2 is F or L. In some embodiments, the polypeptide is an antibody. In some embodiments, the polypeptide comprises the CDR1. In some embodiments, the polypeptide comprises the CDR2. In some embodiments, the polypeptide comprises the CDR3. In some embodiments, the polypeptide comprises the CDR1, the CDR2, and the CDR3. In some embodiments, the polypeptide comprises a light chain variable region comprising CDR1, the CDR2, and/or the CDR3. In some embodiments, the polypeptide further comprises a heavy chain variable region (e.g., a heavy chain variable region described herein).

In some embodiments, the polypeptide comprises light chain CDRs comprising (i) a CDR1 comprising the sequence X.sub.1ASQX.sub.2IRNX.sub.3LN (SEQ ID NO:34), wherein X.sub.1 is S or R, X.sub.2 is G or D, and X.sub.3 is S or N; (ii) a CDR2 comprising the sequence YSSNLX.sub.1X.sub.2 (SEQ ID NO:35), wherein X.sub.1 is H or Q and X.sub.2 is S or T; and (iii) a CDR3 comprising the sequence QQSX.sub.1KLPX.sub.2T (SEQ ID NO:36), wherein X.sub.1 is I or N and X.sub.2 is F or L. In some embodiments, the polypeptide comprises a light chain variable region comprising the light chain CDRs.

In some embodiments, the polypeptide comprises a light chain CDR1 comprising an amino acid sequence selected from the group consisting of RASQDIRNNLN (SEQ ID NO:61), RASQGIRNNLN (SEQ ID NO:62), and SASQDIRNSLN (SEQ ID NO:63). In some embodiments, the polypeptide comprises a light chain CDR2 comprising an amino acid sequence selected from the group consisting of YSSNLHS (SEQ ID NO:64), YSSNLQS (SEQ ID NO:65) and YSSNLHT (SEQ ID NO:66). In some embodiments, the polypeptide comprises a light chain CDR3 comprising an amino acid sequence selected from the group consisting of QQSIKLPLT (SEQ ID NO:67), QQSIKLPFT (SEQ ID NO:68), and QQSNKLPLT (SEQ ID NO:69). In some embodiments, the polypeptide comprises a light chain variable region comprising the light chain CDRs.

In some embodiments, the polypeptide comprises light chain CDRs comprising (a) a CDR1 comprising the sequence RASQDIRNNLN (SEQ ID NO:61), (b) a CDR2 comprising the sequence YSSNLHS (SEQ ID NO:64), and (c) a CDR3 comprising the sequence QQSIKLPLT (SEQ ID NO:67). In some embodiments, the polypeptide comprises light chain CDRs comprising (a) a CDR1 comprising the sequence RASQGIRNNLN (SEQ ID NO:62), (b) a CDR2 comprising the sequence YSSNLQS (SEQ ID NO:65), and (c) a CDR3 comprising the sequence QQSIKLPFT (SEQ ID NO:68). In some other embodiments, the polypeptide comprises light chain CDRs comprising (a) a CDR1 comprising the sequence SASQDIRNSLN (SEQ ID NO:63), (b) a CDR2 comprising the sequence YSSNLHT (SEQ ID NO:66), and (c) a CDR3 comprising the sequence QQSNKLPLT (SEQ ID NO:69). In some embodiments of each of the above, the polypeptide comprises a light chain variable region that comprises the specified CDR1, CDR2, and CDR3.

In some embodiments, the polypeptide comprises heavy chain CDRs comprising (a) a CDR1 comprising the sequence GFSLTTYGVH (SEQ ID NO:55), (b) a CDR2 comprising the sequence VIWGDGRTDYDAAFMS (SEQ ID NO:58), and (c) a CDR3 comprising the sequence NRHDWFDY (SEQ ID NO:60), and/or light chain CDRs comprising (a) a CDR1 comprising the sequence RASQGIRNNLN (SEQ ID NO:62), (b) a CDR2 comprising the sequence YSSNLQS (SEQ ID NO:65), and (c) a CDR3 comprising the sequence QQSIKLPFT (SEQ ID NO:68). In some embodiments, the polypeptide is an antibody.

In some embodiments, the polypeptide comprises a heavy chain CDR1 comprising the sequence GX.sub.1X.sub.2LX.sub.3TYGVH (SEQ ID NO:31), wherein X.sub.1 is F or Y, X.sub.2 is S or T, and X.sub.3 is T, N, or S, a heavy chain CDR2 comprising the sequence VIWGX.sub.1GRTDYDX.sub.2X.sub.3FMS (SEQ ID NO:32), wherein X.sub.1 is G or D, X.sub.2 is A or S, and X.sub.3 is A or S, and/or a heavy chain CDR3 comprising the sequence X.sub.1RHDWFDY (SEQ ID NO:33), wherein X.sub.1 is N or S, and further comprises a light chain CDR1 comprising the sequence X.sub.1ASQX.sub.2IRNX.sub.3LN (SEQ ID NO:34), wherein X.sub.1 is S or R, X.sub.2 is G or D, and X.sub.3 is S or N, a light chain CDR2 comprising the sequence YSSNLX.sub.1X.sub.2 (SEQ ID NO:35), wherein X.sub.1 is H or Q and X.sub.2 is S or T, and/or a light chain CDR3 comprising the sequence QQSX.sub.1KLPX.sub.2T (SEQ ID NO:36), wherein X.sub.1 is I or N and X.sub.2 is F or L. For instance, in some embodiments, the polypeptide comprises the three heavy chain CDRs CDR1, CDR2, and CDR3, as well as the three light chain CDRs CDR1, CDR2, and CDR3.

The invention also encompasses polypeptides comprising one or more CDRs described herein, wherein the polypeptides further comprise one or more FRs described herein.

In some embodiments, the CDRs and/or FRs are in a sequential order.

The invention further provides a heavy chain variable region comprising one or more (e.g., one, two, or three) of the heavy chain CDRs described herein. In some embodiments, the heavy chain variable region further comprises one or more (e.g., one, two, three, or four) of the heavy chain FRs described herein. In addition, the invention also provides a light chain variable region comprising one or more (e.g., one, two, or three) of the light chain CDRs described herein. In some embodiments, the light chain variable region further comprises one or more (e.g., one, two, three, or four) of the light chain FRs described herein.

In another aspect, the invention provides a polypeptide, comprising one or more heavy chain framework regions (FRs) described herein and/or one or more light chain framework regions described herein. In some embodiments, the polypeptide comprises a heavy chain variable region comprising one or more heavy chain FRs described herein and/or a light chain variable region comprising one or more of the light chain FRs described herein.

In some embodiments, the polypeptide comprises one or more (e.g., one, two, three, or four) FRs, wherein each FR comprises an FR of a heavy chain variable region (VH) shown in FIG. 5 as any of the sequences X384, X385, X386, X387, X388, X399, or X420. In some embodiments, the FR of a heavy chain variable region in FIG. 5 is FR1, FR2, FR3, or FR4. In some embodiments, the polypeptide comprises one or more (e.g., one, two, three, or four) FRs, wherein each FR comprises an FR of a light chain variable region (VL) shown in FIG. 3 as any of the sequences X376, X377, X378, X379, X380, X381, or X394. In some embodiments, the FR of a light chain variable region in FIG. 3 is FR1, FR2, FR3, or FR4. Some embodiments include polypeptides (e.g., antibodies) that comprise one or more FRs of a heavy chain variable region (VH) shown in FIG. 5 as any of the sequences X384, X385, X386, X387, X388, X399, or X420 as well as one or more FRs of a light chain variable region shown in shown in FIG. 3 in any of the sequences X376, X377, X378, X379, X380, X381, or X394.

In some embodiments, the invention provides a polypeptide (such as an antibody) which comprises at least one FR that is substantially homologous to at least one FR, at least two FRs, at least three FRs, or at least four FRs of a VH shown in FIG. 5 as any of the sequences X384, X385, X386, X387, X388, X399, or X420 or a VL shown in FIG. 3 in any of the sequences X376, X377, X378, X379, X380, X381, or X394. In some embodiments, one or more FRs substantially homologous to at least one FR, at least two, at least three, or at least four FRs of a VH or VL shown in FIG. 5 or 3 are at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to at least one, at least two, at least three, or at least four FRs of a VH or VL shown in FIG. 5 as any of the sequences X384, X385, X386, X387, X388, X399, or X420 or shown in FIG. 3 as any of the sequences X376, X377, X378, X379, X380, X381, or X394.

In still another aspect, the invention provides a polypeptide comprising: (a) a heavy chain FR1 comprising the sequence EVQLVX.sub.1SGX.sub.2X.sub.3X.sub.4X.sub.5QPGX.sub.6X.sub.7LRLX.sub.8CX.s- ub.9AS (SEQ ID NO:37), wherein X.sub.1 is E or Q, X.sub.2 is A or G, X.sub.3 is G or E, X.sub.4 is L or V, X.sub.5 is V, K, or E, X.sub.6 is G or E, X.sub.7 is T or S, X.sub.8 is T or S, and X.sub.9 is T or K; (b) a heavy chain FR2 comprising the sequence WVRQAPGKGLEWX.sub.1G (SEQ ID NO:38), wherein X.sub.1 is V or M; (c) a heavy chain FR3 comprising the sequence RVTISX.sub.1DX.sub.2SKX.sub.3TX.sub.4YLQX.sub.5NSLRAEDTAVYYCX.su- b.6R (SEQ ID NO:39), wherein X.sub.1 is K or R, X.sub.2 is N or T, X.sub.3 is S or N, X.sub.4 is V or A, X.sub.5 is M or L, and X.sub.6 is V, M, or T; and/or (d) a heavy chain FR4 comprising the sequence WGQGTTVTVSS (SEQ ID NO:40). In some embodiments, the polypeptide comprises the FR1. In some embodiments, the polypeptide comprises the FR2. In some embodiments, the polypeptide comprises the FR3. In some embodiments, the polypeptide comprises the FR1, the FR2, the FR3, and the FR4. In some embodiments, the polypeptide comprises a heavy chain variable region comprising the FR1, the FR2, the FR3, and/or the FR4. In some embodiments, the polypeptide further comprises a light chain variable region.

In some embodiments, the polypeptide comprises a heavy chain FR1 comprising the sequence EVQLVESGAGVKQPGGTLRLTCTAS (SEQ ID NO:70), EVQLVQSGGGVKQPGETLRLTCTAS (SEQ ID NO:71), EVQLVQSGGGLKQPGETLRLSCTAS (SEQ ID NO:72), or EVQLVESGGGVKQPGETLRLTCTAS (SEQ ID NO:73). In other embodiments, the polypeptide comprises a heavy chain FR2 comprising the sequence WVRQAPGKGLEWVG (SEQ ID NO:74) or WVRQAPGKGLEWMG (SEQ ID NO:75). In other embodiments, the polypeptide comprises a heavy chain FR3 comprising the sequence RVTISKDTSKSTVYLQMNSLRAEDTAVYYCMR (SEQ ID NO:76), RVTISKDTSKSTAYLQMNSLRAEDTAVYYCMR (SEQ ID NO:77), or RVTISKDTSKSTAYLQLNSLRAEDTAVYYCTR (SEQ ID NO:78). In some embodiments, the polypeptide comprises a heavy chain FR4 comprising the sequence WGQGTTVTVSS (SEQ ID NO:40).

In some embodiments, the polypeptide comprises heavy chain framework regions comprising (a) an FR1 comprising the sequence EVQLVESGAGVKQPGGTLRLTCTAS (SEQ ID NO:70), (b) an FR2 comprising the sequence WVRQAPGKGLEWVG (SEQ ID NO:74), (c) an FR3 comprising the sequence RVTISKDTSKSTVYLQMNSLRAEDTAVYYCMR (SEQ ID NO:76), and (d) an FR4 comprising the sequence WGQGTTVTVSS (SEQ ID NO:40). In other embodiments, the polypeptide comprises heavy chain framework regions comprising (a) an FR1 comprising the sequence EVQLVQSGGGVKQPGETLRLTCTAS (SEQ ID NO:71), (b) an FR2 comprising the sequence WVRQAPGKGLEWVG (SEQ ID NO:74), (c) an FR3 comprising the sequence RVTISKDTSKSTAYLQMNSLRAEDTAVYYCMR (SEQ ID NO:77), and (d) an FR4 comprising the sequence WGQGTTVTVSS (SEQ ID NO:40). In still other embodiments, the polypeptide comprises heavy chain framework regions comprising (a) an FR1 comprising the sequence EVQLVQSGGGLKQPGETLRLSCTAS (SEQ ID NO:72), (b) an FR2 comprising the sequence WVRQAPGKGLEWMG (SEQ ID NO:75), (c) an FR3 comprising the sequence RVTISKDTSKSTAYLQLNSLRAEDTAVYYCTR (SEQ ID NO:78), and (d) an FR4 comprising the sequence WGQGTTVTVSS (SEQ ID NO:40). In some alternative embodiments, the polypeptide comprises heavy chain framework regions comprising (a) an FR1 comprising the sequence EVQLVESGGGVKQPGETLRLTCTAS (SEQ ID NO:73), (b) an FR2 comprising the sequence WVRQAPGKGLEWVG (SEQ ID NO:74), (c) an FR3 comprising the sequence RVTISKDTSKSTVYLQMNSLRAEDTAVYYCMR (SEQ ID NO:76), and (d) an FR4 comprising the sequence WGQGTTVTVSS (SEQ ID NO:40). In some embodiments, the heavy chain framework regions are contained within a heavy chain variable region.

In a further aspect, the invention provides a polypeptide, comprising (a) a light chain FR1 comprising the sequence X.sub.1IX.sub.2X.sub.3TQSPSSLSX.sub.4X.sub.5X.sub.6GX.sub.7RX.sub.8TIX.su- b.9C (SEQ ID NO:41), wherein X.sub.1 is D or E, X.sub.2 is L or E, X.sub.3 is M or L, X.sub.4 is A or V, X.sub.5 is S or T, X.sub.6 is L, P, or A, X.sub.7 is D or E, X.sub.8 is V or A, and X.sub.9 is T or S; (b) a light chain FR2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 42); (c) a light chain FR3 comprising the sequence GVPXIRFSGSGSGTDFTLTISRLX.sub.2X.sub.3EDX.sub.4AX.sub.5YYC (SEQ ID NO: 43), wherein X.sub.1 is S, D, or A, X.sub.2 is E or Q, X.sub.3 is P or A, X.sub.4 is F or V, and X.sub.5 is T, A, or I; and/or (d) a light chain FR4 comprising the sequence FGSGTKVEIK (SEQ ID NO:44). In some embodiments, the polypeptide comprises the FR1. In some embodiments, the polypeptide comprises the FR2. In some embodiments, the polypeptide comprises the FR3. In some embodiments, the polypeptide comprises the FR4. In some embodiments, the polypeptide comprises the FR1, the FR2, the FR3, and the FR4. In some embodiments, the polypeptide comprises a light chain variable region comprising the FR1, FR2, FR3, and/or FR4. In some embodiments, the polypeptide further comprises a heavy chain variable region.

In some embodiments, the polypeptide comprises a light chain FR1 comprising the sequence DILMTQSPSSLSASPGDRVTISC (SEQ ID NO:79), DILLTQSPSSLSATPGERATITC (SEQ ID NO:80), or EIEMTQSPSSLSVSAGERATISC (SEQ ID NO:81). In some embodiments, the polypeptide comprises a light chain FR2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO:42). In some embodiments, the polypeptide comprises a light chain FR3 comprising the sequence GVPDRFSGSGSGTDFTLTISRLEPEDFAAYYC (SEQ ID NO:82), GVPSRFSGSGSGTDFTLTISRLQPEDVAAYYC (SEQ ID NO:83), or GVPARFSGSGSGTDFTLTISRLEPEDVAIYYC (SEQ ID NO:84). In some embodiments, the polypeptide comprises a light chain FR4 comprising the sequence FGSGTKVEIK (SEQ ID NO:44).

In some embodiments, the polypeptide comprises light chain framework regions comprising (a) an FR1 comprising the sequence DILMTQSPSSLSASPGDRVTISC (SEQ ID NO:79), (b) an FR2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO:42), c) an FR3 comprising the sequence GVPDRFSGSGSGTDFTLTISRLEPEDFAAYYC (SEQ ID NO:82), and (d) an FR4 comprising the sequence FGSGTKVEIK (SEQ ID NO:44). In some embodiments, the polypeptide comprises light chain framework regions comprising (a) an FR1 comprising the sequence DILLTQSPSSLSATPGERATITC (SEQ ID NO:80), (b) an FR2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO:42), (c) an FR3 comprising the sequence GVPSRFSGSGSGTDFTLTISRLQPEDVAAYYC (SEQ ID NO:83), and (d) an FR4 comprising the sequence FGSGTKVEIK (SEQ ID NO:44). In some embodiments, the polypeptide comprises light chain framework regions comprising (a) an FR1 comprising the sequence EIEMTQSPSSLSVSAGERATISC (SEQ ID NO:81), (b) an FR2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO:42), (c) an FR3 comprising the sequence GVPARFSGSGSGTDFTLTISRLEPEDVAIYYC (SEQ ID NO:84), and (d) an FR4 comprising the sequence FGSGTKVEIK (SEQ ID NO:44). In some embodiments, the light chain framework regions are contained within a light chain variable region.

The invention further provides a heavy chain variable region comprising one or more (e.g., one, two, three, or four) of the heavy chain FRs described herein. In addition, the invention also provides a light chain variable region comprising one or more (e.g., one, two, three, or four) of the light chain FRs described herein.

The invention further provides a polypeptide comprising one or more of the heavy chain variable regions described herein and/or one of the light chain variable regions described herein. In some embodiments, the polypeptide comprises a heavy chain variable region comprising one or more of the heavy chain CDRs described herein and/or one or more of the heavy chain FRs described herein. In some embodiments, the polypeptide comprises a light chain variable region comprising one or more of the light chain CDRs described herein and/or one or more of the light chain FRs described herein.

In another aspect, the invention provides a polypeptide comprising the amino acid sequence EVQLVX.sub.1SGX.sub.2X.sub.3X.sub.4X.sub.5QPGX.sub.6X.sub.7LRLX.sub.8CX.s- ub.9ASGX.sub.10X.sub.11LX.sub.12TYGVHWVRQAPGKGLEWX.sub.13GVIWGX.sub.14GRTD- YDX.sub.15X.sub.16FMSRVTISX.sub.17DX.sub.18SKX.sub.19TX.sub.20YLQX.sub.21N- SLRAEDTAVYYCX.sub.22 RX.sub.23RHDWFDYWGQGTTVTVSS (SEQ ID NO:29), wherein X.sub.1 is E or Q, X.sub.2 is A or G, X.sub.3 is G or E, X.sub.4 is L or V, X.sub.5 is V, K, or E, X.sub.6 is G or E, X.sub.7 is T or S, X.sub.8 is T or S, X.sub.9 is T or K, X.sub.10 is F or Y, X.sub.11 is S or T, X.sub.12 is T, N, or S, X.sub.13 is V or M, X.sub.14 is G or D, X.sub.15 is A or S, X.sub.16 is A or S, X.sub.17 is K or R, X.sub.18 is N or T, X.sub.19 is S or N, X.sub.20 is V or A, X.sub.21 is M or L, X.sub.22 is V, M, or T, and X.sub.23 is N or S. In some embodiments, the polypeptide comprises a heavy chain variable region comprising the sequence EVQLVX.sub.1SGX.sub.2X.sub.3X.sub.4X.sub.5QPGX.sub.6X.sub.7LRLX.sub.8CX.s- ub.9ASGX.sub.10X.sub.11LX.sub.12TYGVHWVRQAPGKGLEWX.sub.13GVIWGX.sub.14GRTD- YDX.sub.15X.sub.16FMSRVTISX.sub.17DX.sub.18SKX.sub.19TX.sub.20YLQX.sub.21N- SLRAEDTAVYYCX.sub.22 RX.sub.23RHDWFDYWGQGTTVTVSS (SEQ ID NO:29), wherein X.sub.1 is E or Q, X.sub.2 is A or G, X.sub.3 is G or E, X.sub.4 is L or V, X.sub.5 is V, K, or E, X.sub.6 is G or E, X.sub.7 is T or S, X.sub.8 is T or S, X.sub.9 is T or K, X.sub.10 is F or Y, X.sub.11 is S or T, X.sub.12 is T, N, or S, X.sub.13 is V or M, X.sub.14 is G or D, X.sub.15 is A or S, X.sub.16 is A or S, X.sub.17 is K or R, X.sub.18 is N or T, X.sub.19 is S or N, X.sub.20 is V or A, X.sub.21 is M or L, X.sub.22 is V, M, or T, and X.sub.23 is N or S. In some embodiments, the polypeptide is an antibody. In some embodiments, the polypeptide further comprises a light chain variable region.

The CDRs (CDR1, CDR2, and CDR3) of the heavy chain variable region are underlined (and positioned in sequential order) in the following sequence: EVQLVX.sub.1SGX.sub.2X.sub.3X.sub.4X.sub.5QPGX.sub.6X.sub.7LRLX- .sub.8CX.sub.9ASGX.sub.10X.sub.11LX.sub.12TYGVHWVRQAPGKGLEWX.sub.13GVIWGX.- sub.14GRTDYDX.sub.15X.sub.16FMSRVTISX.sub.17DX.sub.18SKX.sub.19TX.sub.20YL- QX.sub.21NSLRAEDTAVYYCX.sub.22RX.sub.23RHDWFDYWGQGTTVTVSS (SEQ ID NO:29), wherein X.sub.1 is E or Q, X.sub.2 is A or G, X.sub.3 is G or E, X.sub.4 is L or V, X.sub.5 is V, K, or E, X.sub.6 is G or E, X.sub.7 is T or S, X.sub.8 is T or S, X.sub.9 is T or K, X.sub.10 is F or Y, X.sub.11 is S or T, X.sub.12 is T, N, or S, X.sub.13 is V or M, X.sub.14 is G or D, X.sub.15 is A or S, X.sub.16 is A or S, X.sub.17 is K or R, X.sub.18 is N or T, X.sub.19 is S or N, X.sub.20 is V or A, X.sub.21 is M or L, X.sub.22 is V, M, or T, and X.sub.23 is N or S. CDR2 and CDR3 of the heavy chain variable region (CDR-H2 and CDR-H3) were defined according to the Kabat definition (Kabat et al., Sequences of Proteins of Immunological Interest, 4.sup.th edit., National Institutes of Health, Bethesda, Md. (1987)). CDR1 of the heavy chain variable region (CDR-H1) was defined according to a combination of the Kabat and Chothia definitions (Chothia et al., J. Mol. Biol., 196:901-917). Such methods are known in the art for defining antibody CDRs. See, e.g., Chen et al., J. Mol. Biol., 293:865-881 (1999) and Muller et al., Structure, 6:1153-1167 (1998). Within the sequence shown in SEQ ID NO:29, the position of CDR1 is H26-35, the position of CDR2 is H50-65, and the position of CDR3 is H95-102, based on Kabat numbering. According to sequential numbering, the position of CDR1 is H26-35, the position of CDR2 is H50-65, and the position of CDR3 is H98-105. (See, e.g., FIG. 5 for an illustration of the position of the CDRs and FRs of the heavy chain variable region on selected, exemplary heavy chain variable region sequences.)

In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of EVQLVESGAGVKQPGGTLRLTCTASGFSLTTYGVHWVRQAPGKGLEWVGVIWGDGRT DYDAAFMSRVTISKDTSKSTVYLQMNSLRAEDTAVYYCMRNRHDWFDYWGQGTTVT VSS (SEQ ID NO:22; X384), EVQLVQSGGGVKQPGETLRLTCTASGFSLTTYGVHWVRQAPGKGLEWVGVIWGDGRT DYDAAFMSRVTISKDTSKSTAYLQMNSLRAEDTAVYYCMRNRHDWFDYWGQGTTVT VSS (SEQ ID NO:23; X385), EVQLVQSGGGLKQPGETLRLSCTASGYSLTTYGVHWVRQAPGKGLEWMGVIWGDGRT DYDSSFMSRVTISKDTSKSTAYLQLNSLRAEDTAVYYCTRNRHDWFDYWGQGTTVTVS S (SEQ ID NO:26; X388), and EVQLVESGGGVKQPGETLRLTCTASGFSLSTYGVHWVRQAPGKGLEWVGVIWGDGRT DYDAAFMSRVTISKDTSKSTVYLQMNSLRAEDTAVYYCMRNRHDWFDYWGQGTTVT VSS (SEQ ID NO:28; X420). In some embodiments, the polypeptide (e.g., an antibody) comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:22 (X384), SEQ ID NO:23 (X385), SEQ ID NO:26 (X388), and SEQ ID NO:28 (X420).

In an additional aspect, the invention provides a polypeptide comprising the sequence X.sub.1IX.sub.2X.sub.3TQSPSSLSX.sub.4X.sub.5X.sub.6GX.sub.7RX.sub.8TIX.su- b.9CX.sub.10ASQX.sub.11IRNX.sub.12LNWYQQKPGQAPRLLIYYSS NLX.sub.13X.sub.4GVPX.sub.15RFSGSGSGTDFTLTISRLX.sub.16X.sub.17EDX.sub.18A- X.sub.19YYCQQSX.sub.20KLPX.sub.21TFGSGT KVEIK (SEQ ID NO:30), wherein X.sub.1 is D or E, X.sub.2 is L or E, X.sub.3 is M or L, X.sub.4 is A or V, X.sub.5 is S or T, X.sub.6 is L, P, or A, X.sub.7 is D or E, X.sub.8 is V or A, X.sub.9 is T or S, X.sub.10 is S or R, X.sub.11 is G or D, X.sub.12 is S or N, X.sub.13 is H or Q, X.sub.14 is S or T, X.sub.15 is S, D, or A, X.sub.16 is E or Q, X.sub.17 is P or A, X.sub.18 is F or V, X.sub.19 is T, A, or I, X.sub.20 is I or N and X.sub.21 is F or L. In some embodiments, the polypeptide comprises a light chain variable region comprising an amino acid sequence of X.sub.1IX.sub.2X.sub.3TQSPSSLSX.sub.4X.sub.5X.sub.6GX.sub.7RX.sub.8TIX.su- b.9CX.sub.10ASQX.sub.11IRNX.sub.12LNWYQQKPGQAPRLLIYYSS NLX.sub.13X.sub.14GVPX.sub.15RFSGSGSGTDFTLTISRLX.sub.16X.sub.17EDX.sub.18- AX.sub.19YYCQQSX.sub.20KLPX.sub.21TFGSGT KVEIK (SEQ ID NO:30), wherein X.sub.1 is D or E, X.sub.2 is L or E, X.sub.3 is M or L, X.sub.4 is A or V, X.sub.5 is S or T, X.sub.6 is L, P, or A, X.sub.7 is D or E, X.sub.8 is V or A, X.sub.9 is T or S, X.sub.10 is S or R, X.sub.11 is G or D, X.sub.12 is S or N, X.sub.13 is H or Q, X.sub.14 is S or T, X.sub.15 is S, D, or A, X.sub.16 is E or Q, X.sub.17 is P or A, X.sub.18 is F or V, X.sub.19 is T, A, or I, X.sub.20 is I or N and X.sub.21 is F or L. In some embodiments, the polypeptide is an antibody. In some embodiments, the polypeptide further comprises a heavy chain variable region.

The CDRs (CDR1, CDR2, and CDR3) of the light chain variable region are underlined (and positioned in sequential order) in the following sequence: X.sub.1IX.sub.2X.sub.3TQSPSSLSX.sub.4X.sub.5X.sub.6GX.sub.7RX.s- ub.8TIX.sub.9CX.sub.10ASQX.sub.11IRNX.sub.12LNWYQQKPGQAPRLLIYYSS NLX.sub.13X.sub.14GVPX.sub.15RFSGSGSGTDFTLTISRLX.sub.16X.sub.17EDX.sub.18- AX.sub.19YYCQQSX.sub.20KLPX.sub.21TFGSGT KVEIK (SEQ ID NO:30), wherein X.sub.1 is D or E, X.sub.2 is L or E, X.sub.3 is M or L, X.sub.4 is A or V, X.sub.5 is S or T, X.sub.6 is L, P, or A, X.sub.7 is D or E, X.sub.8 is V or A, X.sub.9 is T or S, X.sub.10 is S or R, X.sub.1, is G or D, X.sub.12 is S or N, X.sub.13 is H or Q, X.sub.14 is S or T, X.sub.15 is S, D, or A, X.sub.16 is E or Q, X.sub.17 is P or A, X.sub.18 is F or V, X.sub.19 is T, A, or I, X.sub.20 is I or N and X.sub.21 is F or L. The CDR1, CDR2, and CDR3 of the light chain variable region (CDR-L1, CDR-L2, and CDR-L3, respectively) shown were defined according to the Kabat definition (Kabat et al., Sequences of proteins of Immunological Interest, 4.sup.th edit., National Institutes of Health, Bethesda, Md. (1987)). The position of the CDR1 in the light chain variable region is L24-34, the position of the CDR2 in the light chain variable region is L50-56, and the position of the CDR3 in the light chain variable region is L89-97 (under both sequential and Kabat numbering schemes). (See, e.g., FIG. 3 for an illustration of the position of the CDRs and FRs of the light chain variable region on selected, exemplary light chain variable region sequences.)

In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of -- see Original Patent.

In some embodiments, the polypeptide (e.g., an antibody) comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:15 (X376), SEQ ID NO:18 (X379), and SEQ ID NO:19 (X380).

In some embodiments, the polypeptide comprises both (a) an amino acid sequence selected from the group consisting of SEQ ID NO:22 (X384), SEQ ID NO:23 (X385), SEQ ID NO:26 (X388), and SEQ ID NO:28 (X420) and (b) an amino acid sequence selected from the group consisting of SEQ ID NO:15 (X376), SEQ ID NO:18 (X379), and SEQ ID NO:19 (X380). In some embodiments, the polypeptide (e.g., an antibody) comprises both (a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:22 (X384), SEQ ID NO:23 (X385), SEQ ID NO:26 (X388), and SEQ ID NO:28 (X420) and (b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:15 (X376), SEQ ID NO:18 (X379), and SEQ ID NO:19 (X380).

In some embodiments, the polypeptide comprises an amino acid sequence SEQ ID NO:26 (X388) and an amino acid sequence SEQ ID NO:15 (X376). In some embodiments, the polypeptide comprises an amino acid sequence SEQ ID NO:22 (X384) and an amino acid sequence SEQ ID NO:18 (X379). In some embodiments, the polypeptide comprises an amino acid sequence SEQ ID NO:28 (X420) and an amino acid sequence SEQ ID NO:19 (X380). In some embodiments, the polypeptide comprises an amino acid sequence SEQ ID NO:23 (X385) and an amino acid sequence SEQ ID NO:19 (X380). In some embodiments, the polypeptide is an antibody.

In some embodiments, the polypeptide comprises a heavy chain variable region comprising an amino acid sequence SEQ ID NO:26 (X388) and a light chain variable region comprising an amino acid sequence SEQ ID NO:15 (X376). In some embodiments, the polypeptide comprises a heavy chain variable region comprising an amino acid sequence SEQ ID NO:22 (X384) and a light chain variable region comprising an amino acid sequence SEQ ID NO:18 (X379). In some embodiments, the polypeptide comprises a heavy chain variable region comprising an amino acid sequence SEQ ID NO:28 (X420) and a light chain variable region comprising an amino acid sequence SEQ ID NO:19 (X380). In some embodiments, the polypeptide comprises a heavy chain variable region comprising an amino acid sequence SEQ ID NO:23 (X385) and a light chain variable region comprising an amino acid sequence SEQ ID NO:19 (X380). In some embodiments, the polypeptide comprising the heavy chain variable region and the light chain variable region is an antibody.

The invention provides a polypeptide, such as an antibody, comprising (a) an amino acid sequence having at least about 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:15-21 or to a fragment of an amino acid sequence selected from the group consisting of SEQ ID NOS:15-21, and/or (b) an amino acid sequence having at least about 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:22-28 or to a fragment of an amino acid sequence selected from the group consisting of SEQ ID NOS:22-28. In some embodiments the amino acid sequences have at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 99% identity, or 100% identity to the indicated sequences. In some embodiments, the polypeptide comprises (a) an amino acid sequence having at least about 90% identity to SEQ ID NO:18 or to a fragment of amino acid sequence SEQ ID NO:18, and/or (b) an amino acid sequence having at least about 90% identity to an amino acid sequence SEQ ID NO:22 or to a fragment of to an amino acid sequence SEQ ID NO:22. In some embodiments, the fragment comprises at least about 10 amino acids, at least about 25 amino acids, at least about 50 amino acids, or at least about 100 amino acids. In some embodiments, the polypeptide binds CD26. In some embodiments, the polypeptide is not a murine monoclonal antibody.

In another aspect, the invention provides a polypeptide comprising an amino acid sequence having at least about 80% identity to an amino acid sequence selected from the group consisting of the following sequences shown in FIG. 5: X384, X385, X386, X387, X388, X399, and X420 (SEQ ID NOS:22-28, respectively). The invention also provides a polypeptide, such as an antibody, comprising a heavy chain variable region comprising an amino acid sequence having at least about 80% identity to an amino acid sequence selected from the group consisting of the following sequences shown in FIG. 5: X384, X385, X386, X387, X388, X399, and X420 (SEQ ID NOS:22-28, respectively). For instance, in some embodiments, the heavy chain variable region comprises an amino acid sequence having at least about 80% identity to an amino acid sequence having at least about 80% identity to SEQ ID NO:22 (X384). In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least about 80% identity to SEQ ID NO:23 (X385). In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least about 80% identity to SEQ ID NO:26 (X388). In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least about 80% identity to SEQ ID NO:28 (X420). In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 99% identity, or 100% identity to an amino acid sequence selected from the group consisting of the following sequences shown in FIG. 5: X384, X385, X386, X387, X388, X399, and X420 (SEQ ID NOS:22-28, respectively). For instance, in some embodiments, the heavy chain variable region comprises at least about 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:22-28. In some other embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:22-28.

In a further aspect, the invention provides a polypeptide comprising an amino acid sequence having at least about 80% identity to an amino acid sequence selected from the group consisting of the following sequences shown in FIG. 3: X376, X377, X378, X379, X380, X381, and X394 (SEQ ID NOS:15-21, respectively). The invention further provides a polypeptide, such as an antibody, comprising a light chain variable region comprising an amino acid sequence having at least about 80% identity to an amino acid sequence selected from the group consisting of the following sequences shown in FIG. 3: X376, X377, X378, X379, X380, X381, and X394 (SEQ ID NOS:15-21, respectively). For instance, in some embodiments, the light chain variable region comprises an amino acid sequence having at least about 80% identity to an amino acid sequence of SEQ ID NO:15 (X376). In some embodiments, the light chain variable region comprises an amino acid sequence having at least about 80% identity to an amino acid sequence of SEQ ID NO:18 (X379). In some embodiments, the light chain variable region comprises an amino acid sequence having at least about 80% identity to an amino acid sequence of SEQ ID NO:19 (X380). In some embodiments, the light chain variable region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 99% identity, or 100% identity to an amino acid sequence selected from the group consisting of the following sequences shown in FIG. 3: X376, X377, X378, X379, X380, X381, and X394 (SEQ ID NOS:15-21, respectively). For instance, in some embodiments, the light chain variable region comprises at least about 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:15-21. In other embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:15-21.

In some embodiments, the antibody comprises both a heavy chain variable region comprising an amino acid sequence having at least about 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:22-28 and a light chain variable region comprising an amino acid sequence having at least about 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:15-21. In some embodiments, the antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:15-21 and a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:22-28.

In some embodiments, the polypeptide comprises at least 5 contiguous amino acids, at least 8 contiguous amino acids, at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, at least about 20 contiguous amino acids, at least about 30 contiguous amino acids, or at least about 50 contiguous amino acids of an amino acid sequence of any one of SEQ ID NOS:15-28.

In another aspect, the invention provides a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:15-28.

An E. coli sample containing a plasmid encoding the heavy and light chains of rhuMAb 411 has been deposited under the name "DH5.alpha. Escherichia coli with plasmid having insert of heavy and light chain of a humanized monoclonal antibody against human CD26 cDNA," and with the strain designation "S604069.YST-pABMC148 (x411)," with the American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, Va., 20108, United States of America (10801 University Blvd., Manassas, Va. 20110-2209, United States of America) on Jun. 30, 2006, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of a Patent Procedure, and designated with accession number PTA-7695. Accordingly, the invention provides a polypeptide comprising the heavy chain and/or the light chain of the antibody encoded by the plasmid in E. coli in the sample named "DH5.alpha. Escherichia coli with plasmid having insert of heavy and light chain of a humanized monoclonal antibody against human CD26 cDNA," having strain designation "5604069.YST-pABMC148 (x411)," deposited with the American Type Culture Collection (ATCC) under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of a Patent Procedure. The invention further provides a polypeptide comprising the heavy chain variable region and/or the light chain variable region of the antibody encoded by the plasmid in E. coli in the sample named "DH5.alpha. Escherichia coli with plasmid having insert of heavy and light chain of a humanized monoclonal antibody against human CD26 cDNA," having strain designation "5604069.YST-pABMC148 (x411)," deposited with the ATCC on Jun. 30, 2006, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of a Patent Procedure, and designated with accession number PTA-7695. The invention also provides an antibody encoded by the plasmid deposited with the ATCC in E. coli as accession number PTA-7695.

The invention further provides polypeptides comprising fragments of the polypeptide sequences described herein (e.g., any one of SEQ ID NOS:15-21, SEQ IDS NOS: 22-28, SEQ IDS NO:29, or SEQ IDS NO:30). In some embodiments, the polypeptide comprises a fragment of a polypeptide sequence described herein, wherein the fragment is at least about 10 amino acids in length, at least about 25 amino acids in length, at least about 50 amino acids in length, at least about 75 amino acids in length, or at least about 100 amino acids in length.

The invention further provides a polypeptide (e.g., an antibody) comprising SEQ ID NO:217 (see Example 4, below), or a fragment or variant thereof. In some embodiments, the polypeptide comprises SEQ ID NO:217. In some embodiments, the polypeptide comprises SEQ ID NO:217 except for the signal sequence. (One of ordinary skill in the art will readily appreciate that in some embodiments, the signal sequence of a polypeptide is cleaved off of the polypeptide.) In some embodiments, the polypeptide comprises the variable region of SEQ ID NO:217. In some embodiments, the polypeptide comprises a polypeptide having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% identity to SEQ ID NO:217 (or a fragment thereof). In some embodiments, the polypeptide comprises a fragment of SEQ ID NO:217 (or of its variable region), wherein the fragment is at least about 10 amino acids in length, at least about 25 amino acids in length, at least about 50 amino acids in length, at least about 75 amino acids in length, or at least about 100 amino acids in length. In some embodiments, the polypeptide binds human CD26.

The invention further provides a polypeptide (e.g., an antibody) comprising SEQ ID NO:218 (see Example 4, below), or a fragment or variant thereof. In some embodiments, the polypeptide comprises SEQ ID NO:218. In some embodiments, the polypeptide comprises SEQ ID NO:218 except for the signal sequence. (One of ordinary skill in the art will readily appreciate that in some embodiments, the signal sequence of a polypeptide is cleaved off of the polypeptide.) In some embodiments, the polypeptide comprises the variable region of SEQ ID NO:218. In some embodiments, the polypeptide comprises a polypeptide having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% identity to SEQ ID NO:218 (or a fragment thereof). In some embodiments, the polypeptide comprises a fragment of SEQ ID NO:218 (or of its variable region), wherein the fragment is at least about 10 amino acids in length, at least about 25 amino acids in length, at least about 50 amino acids in length, at least about 75 amino acids in length, or at least about 100 amino acids in length. In some embodiments, the polypeptide further comprises SEQ ID NO:218 (see Example 4, below), or a fragment or variant thereof. In some embodiments, the polypeptide binds human CD26. For instance, in some embodiments, the polypeptide is an antibody comprising at least one heavy chain (e.g., two heavy chains), each of which comprises SEQ ID NO:217 without the signal sequence, and at least one light chain (e.g., two light chains), each of which comprises SEQ ID NO:218 without the signal sequence.

The invention further provides a polypeptide comprising the polypeptide of SEQ ID NO:219 and/or SEQ ID NO:220 (shown in Example 13, below), or a fragment thereof, or a variant thereof.

In another aspect, the invention provides a polypeptide, such as an antibody, that binds to one or more peptides selected from the group consisting of YSLRWISDHEYLY (SEQ ID NO:45; peptide 6), LEYNYVKQWRHSY (SEQ ID NO:46; peptide 35), TWSPVGHKLAYVW (SEQ ID NO:47; peptide 55), LWWSPNGTFLAYA (SEQ ID NO:48; peptide 84), RISLQWLRRIQNY (SEQ ID NO:49; peptide 132), YVKQWRHSYTASY (SEQ ID NO:50; peptide 37), EEEVFSAYSALWW (SEQ ID NO:51; peptide 79), DYSISPDGQFILL (SEQ ID NO:52; peptide 29), SISPDGQFILLEY (SEQ ID NO:53; peptide 30), and IYVKIEPNLPSYR (SEQ ID NO:54; peptide 63). In some embodiments, the polypeptide preferentially binds to the one or more peptides. These peptides are regions of human CD26. In some embodiments, the polypeptide preferentially binds to one or more peptides selected from the group consisting of YSLRWISDHEYLY (SEQ ID NO:45; peptide 6), LEYNYVKQWRHSY (SEQ ID NO:46; peptide 35), TWSPVGHKLAYVW (SEQ ID NO:47; peptide 55), LWWSPNGTFLAYA (SEQ ID NO:48; peptide 84), RISLQWLRRIQNY (SEQ ID NO:49; peptide 132), YVKQWRHSYTASY (SEQ ID NO:50; peptide 37), EEEVFSAYSALWW (SEQ ID NO:51; peptide 79), DYSISPDGQFILL (SEQ ID NO:52; peptide 29), SISPDGQFILLEY (SEQ ID NO:53; peptide 30), and IYVKIEPNLPSYR (SEQ ID NO:54; peptide 63), relative to one or more peptides corresponding to other regions of human CD26.

In some embodiments, the polypeptide (e.g., antibody) binds to each of the following peptides: YSLRWISDHEYLY (SEQ ID NO:45; peptide 6); LEYNYVKQWRHSY (SEQ ID NO:46; peptide 35); TWSPVGHKLAYVW (SEQ ID NO:47; peptide 55); LWWSPNGTFLAYA (SEQ ID NO:48; peptide 84); and RISLQWLRRIQNY (SEQ ID NO:49; peptide 132). In some other embodiments, the polypeptide binds to each of the following peptides: YSLRWISDHEYLY (SEQ ID NO:45; peptide 6); TWSPVGHKLAYVW (SEQ ID NO:47; peptide 55); RISLQWLRRIQNY (SEQ ID NO:49; peptide 132); YVKQWRHSYTASY (SEQ ID NO:50; peptide 37); and EEEVFSAYSALWW (SEQ ID NO:51; peptide 79). In some embodiments, the polypeptide binds to each of the following peptides: DYSISPDGQFILL (SEQ ID NO:52; peptide 29); SISPDGQFILLEY (SEQ ID NO:53; peptide 30); and TWSPVGHKLAYVW (SEQ ID NO:47; peptide 55). In some other embodiments, the polypeptide binds to each of the following peptides: DYSISPDGQFILL (SEQ ID NO:52; peptide 29); SISPDGQFILLEY (SEQ ID NO:53; peptide 30); TWSPVGHKLAYVW (SEQ ID NO:47; peptide 55); and IYVKIEPNLPSYR (SEQ ID NO:54; peptide 63). In some embodiments, the polypeptides preferentially bind to the specified peptides.

Competition assays can be used to determine whether two antibodies bind the same epitope by recognizing identical or sterically overlapping epitopes. See, e.g., Dong et al. (1998) and the specificity experiments in the Examples below. Typically, antigen is immobilized on a multi-well plate and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured. Common labels for such competition assays are radioactive labels or enzyme labels. In addition, epitope mapping techniques known to those in the art can be used to determine the epitopes with which antibodies bind. See, e.g., Example 3(e) below.

In some embodiments, a polypeptide described herein comprises one or more constant regions. In some embodiments, a polypeptide described herein comprises a human constant region. In some embodiments, the constant region is a constant region of the heavy chain. In other embodiments, the constant region is a constant region of the light chain. In some embodiments, the polypeptide comprises a constant region which has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% identity to a human constant region. In some embodiments, a polypeptide (e.g., an antibody) described herein comprises an Fc region. In some embodiments, the polypeptide comprises a human Fc region. In some embodiments, a polypeptide described herein comprises an Fc region which has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% identity to a human Fc region.

In some embodiments, an antibody described herein is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some other embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is a human IgG antibody.

The invention provides antibodies in monomeric, dimeric and multivalent forms. For example, bispecific antibodies, monoclonal antibodies that have binding specificities for at least two different antigens, can be prepared using the antibodies disclosed herein. Methods for making bispecific antibodies are known in the art (see, e.g., Suresh et al., 1986, Methods in Enzymology 121:210). Traditionally, the recombinant production of bispecific antibodies was based on the coexpression of two immunoglobulin heavy chain-light chain pairs, with the two heavy chains having different specificities (Millstein and Cuello, 1983, Nature 305, 537-539).

According to one approach to making bispecific antibodies, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2 and CH3 regions. It is preferred to have the first heavy chain constant region (CH1), containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are cotransfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.

In one approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure, with an immunoglobulin light chain in only one half of the bispecific molecule, facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations. This approach is described in PCT Publication No. WO 94/04690, published Mar. 3, 1994.

Heteroconjugate antibodies, comprising two covalently joined antibodies, are also within the scope of the invention. Such antibodies have been used to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (PCT application publication Nos. WO 91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents and techniques are well known in the art, and are described in U.S. Pat. No. 4,676,980.

In certain embodiments, an antibody described herein is an antibody fragment. For instance, in some embodiments, the antibody is selected from the group consisting of Fab, Fab', Fab'-SH, Fv, scFv and F(ab').sub.2. In some embodiments, the antibody is a Fab. Various techniques have been developed for the production of antibody fragments. These fragments can be derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., 1992, J. Biochem. Biophys. Methods 24:107-117 and Brennan et al., 1985, Science 229:81), or produced directly by recombinant host cells. For example, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab').sub.2 fragments (Carter et al., 1992, Bio/Technology 10:163-167). In another embodiment, the F(ab').sub.2 is formed using the leucine zipper GCN4 to promote assembly of the F(ab').sub.2 molecule. According to another approach, Fv, Fab or F(ab').sub.2 fragments are isolated directly from recombinant host cell culture.

In some embodiments, the antibodies of the invention are single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies linear antibodies, single chain antibodies, and any other modified configuration of the immunoglobulin molecule.

Single chain variable region fragments are made by linking light and/or heavy chain variable regions by using a short linking peptide. Bird et al. (1988) Science 242:423-426. An example of a linking peptide is (GGGGS).sub.3 (SEQ ID NO:85), which bridges approximately 3.5 nm between the carboxy terminus of one variable region and the amino terminus of the other variable region. Linkers of other sequences have been designed and used. Bird et al. (1988). Linkers can in turn be modified for additional functions, such as attachment of drugs or attachment to solid supports. The single chain variants can be produced either recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing polynucleotide that encodes the scFv can be introduced into a suitable host cell, either eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic, such as E. coli. Polynucleotides encoding the scFv of interest can be made by routine manipulations such as ligation of polynucleotides. The resultant scFv can be isolated using standard protein purification techniques known in the art.

Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).

The invention encompasses modifications to antibodies or other polypeptides described herein, including functionally equivalent antibodies which do not significantly affect their properties and variants which have enhanced or decreased activity. It is understood that the principles of modification apply to polypeptides as well as antibodies. Modification of polypeptides is routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids which do not significantly deleteriously change the functional activity, or use of chemical analogs.

Amino acid sequence insertions or additions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the serum half-life of the antibody.

Substitution variants have at least one amino acid residue in the antibody or other polypeptide sequence removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include the CDRs, but FR alterations are also contemplated. Conservative substitutions are shown in Table 1 (see Original Patent) under the heading of "conservative substitutions". If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions" in Table 1 (see Original Patent), or as further described below in reference to amino acid classes, may be introduced and the products screened.

Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr; (3) Acidic: Asp, Glu; (4) Basic: Asn, Gln, His, Lys, Arg; (5) Residues that influence chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions are made by exchanging a member of one of these classes for another class. More conservative substitutions involve exchanging one member of a class for another member of the same class.

Any cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability, particularly where the antibody is an antibody fragment such as an Fv fragment.

Amino acid modifications can range from changing or modifying one or more amino acids to complete redesign of a region, such as the variable region. Changes in the variable region can alter binding affinity and/or specificity. In some embodiments, no more than one to five conservative amino acid substitutions are made within a CDR domain. In other embodiments, no more than one to three conservative amino acid substitutions are made within a CDR3 domain. In still other embodiments, the CDR domain is CDRH3 and/or CDR L3.

Modifications also include glycosylated and nonglycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation. Antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund, 1997, Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32). The oligosaccharide side chains of the immunoglobulins affect the protein's function (Boyd et al., 1996, Mol. Immunol. 32:1311-1318; Wittwe and Howard, 1990, Biochem. 29:4175-4180) and the intramolecular interaction between portions of the glycoprotein, which can affect the conformation and presented three-dimensional surface of the glycoprotein (Hefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech. 7:409-416). Oligosaccharides may also serve to target a given glycoprotein to certain molecules based upon specific recognition structures. Glycosylation of antibodies has also been reported to affect antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO cells with tetracycline-regulated expression of .beta.(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase catalyzing formation of bisecting GlcNAc, was reported to have improved ADCC activity (Umana et al., 1999, Nature Biotech. 17:176-180).

Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

The glycosylation pattern of antibodies may also be altered without altering the underlying nucleotide sequence. Glycosylation largely depends on the host cell used to express the antibody. Since the cell type used for expression of recombinant glycoproteins, e.g. antibodies, as potential therapeutics is rarely the native cell, variations in the glycosylation pattern of the antibodies can be expected (see, e.g. Hse et al., 1997, J. Biol. Chem. 272:9062-9070).

In addition to the choice of host cells, factors that affect glycosylation during recombinant production of antibodies include growth mode, media formulation, culture density, oxygenation, pH, purification schemes and the like. Various methods have been proposed to alter the glycosylation pattern achieved in a particular host organism including introducing or overexpressing certain enzymes involved in oligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and 5,278,299). Glycosylation, or certain types of glycosylation, can be enzymatically removed from the glycoprotein, for example using endoglycosidase H (Endo H). In addition, the recombinant host cell can be genetically engineered to be defective in processing certain types of polysaccharides. These and similar techniques are well known in the art.

Other methods of modification include using coupling techniques known in the art, including, but not limited to, enzymatic means, oxidative substitution and chelation. Modifications can be used, for example, for attachment of labels for immunoassay. Modified polypeptides are made using established procedures in the art and can be screened using standard assays known in the art, some of which are described below and in the Examples.

Other antibody modifications include antibodies that have been modified as described in PCT Publication No. WO 99/58572, published Nov. 18, 1999. These antibodies comprise, in addition to a binding domain directed at the target molecule, an effector domain having an amino acid sequence substantially homologous to all or part of a constant domain of a human immunoglobulin heavy chain. These antibodies are capable of binding the target molecule without triggering significant complement dependent lysis, or cell-mediated destruction of the target. In some embodiments, the effector domain is capable of specifically binding FcRn and/or Fc.gamma.RIIb. These are typically based on chimeric domains derived from two or more human immunoglobulin heavy chain C.sub.H2 domains. Antibodies modified in this manner are particularly suitable for use in chronic antibody therapy, to avoid inflammatory and other adverse reactions to conventional antibody therapy.

In some embodiments, the polypeptides of the invention are conjugates. For instance, in some embodiments, the polypeptide is conjugated to another agent such as a chemotherapeutic agent, a radionuclide, an immunotherapeutic agent, a cytokine, a chemokine, an imaging agent, a toxin, a biological agent, an enzyme inhibitor, or an antibody.

In some embodiments the polypeptides, such as antibodies, are conjugated to water-soluble polymer moieties. The polypeptides may be conjugated to polyethylene glycol (PEG), monomethoxy-PEG, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol or the like. The polypeptides may be modified at random positions with the molecule, or at predetermined positions with the molecule and may include one, two, three or more attached moieties. The polymer may be of any molecular weight, and may be branched or unbranched. In some embodiments, the moiety is attached to the polypeptide via a linker. In some embodiments, the attached moiety increases the circulating half-life of the polypeptide in an animal. Methods of attaching polymers such as PEG to polypeptides including antibodies are well known in the art. In some embodiments, the polypeptides are PEGylated polypeptides, such as PEGylated antibodies.

The invention includes affinity matured embodiments. For example, affinity matured antibodies can be produced by procedures known in the art (Marks et al., 1992, Bio/Technology, 10:779-783; Barbas et al., 1994, Proc Nat. Acad. Sci, USA 91:3809-3813; Schier et al., 1995, Gene, 169:147-155; Yelton et al., 1995, J. Immunol., 155:1994-2004; Jackson et al., 1995, J. Immunol., 154(7):3310-9; Hawkins et al, 1992, J. Mol. Biol., 226:889-896; and WO2004/058184). Candidate affinity matured antibodies may be screened or selected for improved and/or altered binding affinity using any method known in the art, including screening using BIAcore surface plasmon resonance analysis, and selection using any method known in the art for selection, including phage display, yeast display, and ribosome display.

Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, 1975, Nature 256:495. In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

The monoclonal antibodies (as well as other polypeptides) of the invention may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. Methods of preparing polypeptides and monoclonal antibodies are well known in the art. In some embodiments, DNA encoding the monoclonal antibodies is isolated, sequenced, and/or amplified using conventional procedures, such as by using oligonucleotide probes or primers that are capable of binding specifically to genes or polynucleotides encoding the heavy and light chains of the monoclonal antibodies. Sequences encoding polypeptides, such as antibodies, of a desired sequence may also readily be prepared using a combination of synthetic DNA methods, PCR methods, and recombinant techniques well known in the art. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.

In some embodiments, the polypeptides of the invention (e.g., antibodies) are expressed in any organism, or cells derived from any organism, including, but not limited to bacteria, yeast, plant, insect, and mammal. Particular types of cells include, but are not limited to, Drosophila melanogaster cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, SF9 cells, HEK-293 cells, Neurospora, BHK cells, CHO cells, COS cells, HeLa cells, fibroblasts, Schwannoma cell lines, immortalized mammalian myeloid and lymphoid cell lines, Jurkat cells, mast cells and other endocrine and exocrine cells, and neuronal cells.

A variety of protein expression systems, vectors, and cell media useful in the production of polypeptides are known to those of ordinary skill in the art. See, e.g., International Patent Publication Nos. WO 03/054172, WO 04/009823, and WO 03/064630, as well as U.S. Patent Publication Nos. US 2005/0170454, US 2005/0084928, and US 2006/0003405, each of which is incorporated herein by reference in its entirety. In some embodiments, a glutamine synthetase (GS) expression system is used for expression of the polypeptides (e.g., antibodies).

The polypeptide may be purified or isolated after expression according to methods known to those skilled in the art. Examples of purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic affinity, and reverse-phase HPLC chromatography, and chromatofocusing. The degree of purification necessary will vary depending on the use of the polypeptide. In some instances, no purification will be necessary.

The DNA can be modified, for example, by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In that manner, "chimeric" or "hybrid" antibodies are prepared that have the binding specificity of a monoclonal antibody disclosed herein. Typically such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody of the invention, or they are substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for one surface epitope CD26 and another antigen-combining site having specificity for a different antigen or CD26 epitope.

The invention also encompasses humanized antibodies. Therapeutic antibodies often elicit adverse effects, in part due to triggering of an immune response directed against the administered antibody. This can result in reduced drug efficacy, depletion of cells bearing the target antigen, and an undesirable inflammatory response. To circumvent the above, recombinant anti-CD26 humanized antibodies may be generated. The general principle in humanizing an antibody involves retaining the basic sequence of the antigen-binding portion of the antibody, while swapping at least portions of the non-human remainder of the antibody with human antibody sequences. Four traditional, but non-limiting, general steps to humanize a monoclonal antibody include: (1) determining the nucleotide and predicted amino acid sequence of the starting antibody light and heavy variable domains (2) designing the humanized antibody, i.e., deciding which antibody framework region or residues and/or CDR residues to use during the humanizing process (3) the actual humanizing methodologies/techniques and (4) the transfection and expression of the humanized antibody. The constant region may also be engineered to more resemble human constant regions to avoid immune response if the antibody is used in clinical trials and treatments in humans. See, for example, U.S. Pat. Nos. 5,997,867 and 5,866,692.

In the recombinant humanized antibodies, the Fc.gamma. portion can be modified to avoid interaction with Fc.gamma. receptor and the complement immune system. Techniques for preparation of such antibodies are described in WO 99/58572.

A number of "humanized" antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin have been described, including chimeric antibodies having rodent V regions and their associated complementarity determining regions (CDRs) fused to human constant domains. See, for example, Winter et al. Nature 349:293-299 (1991); Lobuglio et al. Proc. Nat. Acad. Sci. USA 86:4220-4224 (1989); Shaw et al. J Immunol. 138:4534-4538 (1987); and Brown et al. Cancer Res. 47:3577-3583 (1987). Other references describe rodent CDRs grafted into a human supporting framework region (FR) prior to fusion with an appropriate human antibody constant domain. See, for example, Riechmann et al. Nature 332:323-327 (1988); Verhoeyen et al. Science 239:1534-1536 (1988); and Jones et al. Nature 321:522-525 (1986). Another reference describes rodent CDRs supported by recombinantly veneered rodent framework regions. See, for example, European Patent Publication No. 519,596. These types of "humanized" molecules are designed to minimize unwanted immunological response toward rodent antihuman antibody molecules which limits the duration and effectiveness of therapeutic applications of those moieties in human recipients. Other methods of humanizing antibodies that may also be utilized are disclosed by Daugherty et al., Nucl. Acids Res., 19:2471-2476 (1991) and in U.S. Pat. Nos. 6,180,377; 6,054,297; 5,997,867; 5,866,692; 6,210,671; 6,350,861; and PCT WO 01/27160.

Additional exemplary methods of humanizing antibodies are described in International Publication No. WO 02/084277 and U.S. Publication No. US 2004/0133357, both of which are incorporated by reference herein in their entirety.

In yet another alternative, fully human antibodies may be obtained by using commercially available mice which have been engineered to express specific human immunoglobulin proteins. Transgenic animals which are designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response may also be used for generation of humanized or human antibodies. Examples of such technology are Xenomouse.TM. from Abgenix, Inc. (Fremont, Calif.) and HuMAb-Mouse.RTM. and TC Mouse.TM. from Medarex, Inc. (Princeton, N.J.).

In another alternative, antibodies may be made recombinantly by phage display technology. See, for example, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743 and 6,265,150; and Winter et al., Annu. Rev. Immunol. 12:433-455 (1994). Alternatively, the phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats; for review see, e.g., Johnson, Kevin S, and Chiswell, David J., Current Opinion in Structural Biology 3, 564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Mark et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). In a natural immune response, antibody genes accumulate mutations at a high rate (somatic hypermutation). Some of the changes introduced will confer higher affinity, and B cells displaying high-affinity surface immunoglobulin are preferentially replicated and differentiated during subsequent antigen challenge. This natural process can be mimicked by employing the technique known as "chain shuffling." Marks, et al., Bio/Technol. 10:779-783 (1992)). In this method, the affinity of "primary" human antibodies obtained by phage display can be improved by sequentially replacing the heavy and light chain V region genes with repertoires of naturally occurring variants (repertoires) of V domain genes obtained from unimmunized donors. This technique allows the production of antibodies and antibody fragments with affinities in the pM-nM range. A strategy for making very large phage antibody repertoires (also known as "the mother-of-all libraries") has been described by Waterhouse et al., Nucl. Acids Res. 21:2265-2266 (1993). Gene shuffling can also be used to derive human antibodies from rodent antibodies, where the human antibody has similar affinities and specificities to the starting rodent antibody. According to this method, which is also referred to as "epitope imprinting", the heavy or light chain V domain gene of rodent antibodies obtained by phage display technique is replaced with a repertoire of human V domain genes, creating rodent-human chimeras. Selection on antigen results in isolation of human variable regions capable of restoring a functional antigen-binding site, i.e., the epitope governs (imprints) the choice of partner. When the process is repeated in order to replace the remaining rodent V domain, a human antibody is obtained (see PCT patent application PCT WO 9306213, published Apr. 1, 1993). Unlike traditional humanization of rodent antibodies by CDR grafting, this technique provides completely human antibodies, which have no framework or CDR residues of rodent origin. It is apparent that although the above discussion pertains to humanized antibodies, the general principles discussed are applicable to customizing antibodies for use, for example, in dogs, cats, primates, equines and bovines.

Chimeric or hybrid antibodies also may be prepared in vitro using known methods of synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

Single chain Fv fragments may also be produced, such as described in Iliades et al., 1997, FEBS Letters, 409:437-441. Coupling of such single chain fragments using various linkers is described in Kortt et al., 1997, Protein Engineering, 10:423-433. A variety of techniques for the recombinant production and manipulation of antibodies are well known in the art.

In one aspect, the invention provides methods of producing the polypeptides described herein. In some embodiments, the method comprises expressing a polynucleotide in a host cell, wherein the polynucleotide encodes the polypeptide. In some embodiments, the method is a method of producing an antibody and comprises expressing one or more polynucleotides in a host cell (e.g., in cell culture), wherein each chain of the antibody is encoded by at least one of the polynucleotides. In some embodiments, the one or more polynucleotides are on the same vector. In other embodiments, the one or more polynucleotides are located on separate vectors. In some embodiments, the methods of producing polypeptides described herein further comprise the step of isolating the polypeptides from the host cells in which they are expressed (e.g., isolated from the cell culture in which the host cells are grown).

Polynucleotides, Variant Sequences, Vectors, and Host Cells

The invention further provides polynucleotides encoding the polypeptides, such as antibodies, described herein. In some embodiments, the polynucleotides encode a polypeptide comprising a heavy chain variable region or light chain variable region described herein. Vectors, such as expression vectors, comprising the polynucleotides are also provided. Host cells comprising the vectors and/or polynucleotides of the invention are further provided. In some embodiments, the polynucleotides are isolated.

In one aspect, the invention provides a polynucleotide comprising a nucleic acid sequence selected from the group consisting of each of the sequences shown in FIG. 2 and FIG. 4 (SEQ IDS NOS:1-14), or a fragment thereof. The invention further provides a polynucleotide that hybridizes under moderately stringent conditions to a polynucleotide selected from the group consisting of those polynucleotides shown in FIG. 2 and FIG. 4 (SEQ IDS NOS:1-14). In some embodiments, the polynucleotide hybridizes under highly stringent conditions to a polynucleotide shown in FIG. 2 and FIG. 4 (SEQ IDS NOS:1-14).

In another aspect, the invention provides a polynucleotide comprising a polynucleotide encoding an amino acid sequence selected from the group consisting of each of the following sequences shown in FIG. 3 or FIG. 5: X376, X377, X378, X379, X380, X381, X394, X384, X385, X386, X387, X388, X399, and X420 (SEQ ID NOS:15-28).

The invention further provides a polynucleotide comprising a polynucleotide of the sequence SEQ ID NO:215 and/or SEQ ID NO:216 (see Example 4, below), or a fragment thereof. In some embodiments, the fragment comprises at least 12, at least 20, at least 50, at least 75, or at least 100 nucleotides. In some embodiments, the polynucleotide comprises a polynucleotide comprising the sequence of SEQ ID NO:215 that encodes the heavy chain. In some embodiments, the polynucleotide comprises a polynucleotide comprising the sequence of SEQ ID NO:216 that encodes the light chain. In some embodiments, the polynucleotide comprises the fragment of the sequence of SEQ ID NO:215 that encodes the heavy chain. In some embodiments, the polynucleotide comprises a polynucleotide that comprises the fragment of SEQ ID NO:216 that encodes the light chain. In some embodiments, the polynucleotide comprises the fragment of the sequence of SEQ ID NO:215 that encodes the heavy chain variable region. In some embodiments, the polynucleotide comprises a polynucleotide that comprises the fragment of SEQ ID NO:216 that encodes the light chain variable region.

The invention also provides a polynucleotide (e.g., an antibody) encoding a polypeptide comprising the heavy chain and/or the light chain of the antibody encoded by the plasmid in E. coli in the sample named "DH5.alpha. Escherichia coli with plasmid having insert of heavy and light chain of a humanized monoclonal antibody against human CD26 cDNA," having strain designation "S604069.YST-pABMC148 (x411)," deposited with the American Type Culture Collection (ATCC) on Jun. 30, 2006, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of a Patent Procedure, and designated with accession number PTA-7695. The invention further provides a polynucleotide encoding a polypeptide comprising the heavy chain variable region and/or the light chain variable region of the antibody encoded by the plasmid in E. coli in the sample named "DH5 .alpha. Escherichia coli with plasmid having insert of heavy and light chain of a humanized monoclonal antibody against human CD26 cDNA," having strain designation "S604069.YST-pABMC148 (x411)," deposited with the ATCC on Jun. 30, 2006, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of a Patent Procedure, and designated with accession number PTA-7695. In some embodiments, the polynucleotide comprises the heavy and/or light chain coding sequences of the deposited sequence.

Expression systems comprising expression vectors and host cells can be used in a method of producing a polypeptide, such as an antibody, of the invention, wherein the host cell is cultured and the polypeptide produced by the cultured host cell is recovered. Polynucleotides encoding antibodies of the invention can also be delivered to a host subject for expression of the antibody by cells of the host subject.

Polynucleotides complementary to any such sequences are also encompassed by the present invention. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.

Variants of the polynucleotides and polypeptides described herein are also provided. The polynucleotide and polypeptide variants may contain one or more substitutions, additions, deletions and/or insertions. In some embodiments, the variants are such that the binding of the encoded polypeptide to human CD26 is not substantially diminished, relative to the polypeptide encoded by the original polynucleotide or the polypeptide. The effect on the immunoreactivity of the encoded polypeptide may generally be assessed as described herein. Variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity and most preferably at least about 90% identity to the original sequence or a portion thereof. In some embodiments, where a sequence is being compared to a CDR or a small fragment, the comparison window may be smaller, e.g., at least 7 amino acids or at least 10 amino acids.

Two polynucleotide or polypeptide sequences are said to be "identical" or have "identity" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins--Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E. W. and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA 80:726-730.

Alternatively, the % (amino acid) identity may be obtained using one of the publicly available BLAST or BLAST-2 programs. The WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology 266:460-480 (1996)). Percent (amino acid) sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The BLAST program is based on the alignment method of Karlin and Altschul. Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin And Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997).

In some embodiments, the "percentage of identity" is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e. gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity. In some embodiments, the comparison window may be smaller (e.g., 7 or 10 amino acids).

Variants may also, or alternatively, be substantially homologous to a polynucleotide described herein, or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a native antibody (or a complementary sequence). In some embodiments, the variants are capable of hybridizing under highly stringent conditions to the original sequence.

Suitable "moderately stringent conditions" include prewashing in a solution of 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50.degree. C.-65.degree. C., 5.times.SSC, overnight; followed by washing twice at 65.degree. C. for 20 minutes with each of 2.times., 0.5.times. and 0.2.times.SSC containing 0.1% SDS.

As used herein, "highly stringent conditions" or "high stringency conditions" are those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50.degree. C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42.degree. C.; or (3) employ 50% formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5.times.Denhardt's solution, sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42.degree. C., with washes at 42.degree. C. in 0.2.times.SSC (sodium chloride/sodium citrate) and 50% formamide at 55.degree. C., followed by a high-stringency wash consisting of 0.1.times.SSC containing EDTA at 55.degree. C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention.

Polynucleotides may be prepared using any of a variety of techniques known in the art. DNA encoding an antibody may be obtained from a cDNA library prepared from tissue expressing antibody mRNA. The antibody-encoding gene may also be obtained from a genomic library or by oligonucleotide synthesis. Libraries can be screened with probes (such as binding partners or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Illustrative libraries include human liver cDNA library (human liver 5' stretch plus cDNA, Clontech Laboratories, Inc.) and mouse kidney cDNA library (mouse kidney 5'-stretch cDNA, Clontech laboratories, Inc.). Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989. Alternatively, one can isolate the gene encoding antibody using PCR methodology (Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1995).

Polynucleotide variants may generally be prepared by any method known in the art, including chemical synthesis by, for example, solid phase phosphoramidite chemical synthesis. Modifications in a polynucleotide sequence may also be introduced using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis (see Adelman et al., DNA 2:183, 1983). Alternatively, RNA molecules may be generated by in vitro or in vivo transcription of DNA sequences encoding an antibody, or portion thereof, provided that the DNA is incorporated into a vector with a suitable RNA polymerase promoter (such as T7 or SP6). Certain portions may be used to prepare an encoded polypeptide, as described herein. In addition, or alternatively, a portion may be administered to a patient such that the encoded polypeptide is generated in vivo (e.g., by transfecting antigen-presenting cells, such as dendritic cells, with a cDNA construct encoding the polypeptide, and administering the transfected cells to the patient).

Any polynucleotide may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl-, methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.

Nucleotide sequences can be joined to a variety of other nucleotide sequences using established recombinant DNA techniques. For example, a polynucleotide may be cloned into any of a variety of cloning vectors, including plasmids, phagemids, lambda phage derivatives and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors. In general, a vector will contain an origin of replication functional in at least one organism, convenient restriction endonuclease sites and one or more selectable markers. Other elements will depend upon the desired use, and will be apparent to those of ordinary skill in the art.

Within certain embodiments, polynucleotides may be formulated so as to permit entry into a cell of a mammal, and to permit expression therein. Such formulations are particularly useful for therapeutic purposes, as described below. Those of ordinary skill in the art will appreciate that there are many ways to achieve expression of a polynucleotide in a target cell, and any suitable method may be employed. For example, a polynucleotide may be incorporated into a viral vector such as, but not limited to, adenovirus, adeno-associated virus, retrovirus, or vaccinia or other pox virus (e.g., avian pox virus). Techniques for incorporating DNA into such vectors are well known to those of ordinary skill in the art. A retroviral vector may additionally transfer or incorporate a gene for a selectable marker (to aid in the identification or selection of transduced cells) and/or a targeting moiety, such as a gene that encodes a ligand for a receptor on a specific target cell, to render the vector target specific. Targeting may also be accomplished using an antibody, by methods known to those of ordinary skill in the art.
 

Claim 1 of 2 Claims

1. An isolated polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS:1-14.
 

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