WO2008005828A2 - PHARMACEUTICALLY ACCEPTABLE COMPOSITIONS COMPRISING ANTIBODY MOLECULES SPECIFIC TO LAMININ-5 α3 CHAIN DOMAINS G1G2 AND USE THEREOF - Google PatentsPHARMACEUTICALLY ACCEPTABLE COMPOSITIONS COMPRISING ANTIBODY MOLECULES SPECIFIC TO LAMININ-5 α3 CHAIN DOMAINS G1G2 AND USE THEREOF Download PDF
- Publication number
- WO2008005828A2 WO2008005828A2 PCT/US2007/072457 US2007072457W WO2008005828A2 WO 2008005828 A2 WO2008005828 A2 WO 2008005828A2 US 2007072457 W US2007072457 W US 2007072457W WO 2008005828 A2 WO2008005828 A2 WO 2008005828A2
- WIPO (PCT)
- Prior art keywords
- antibody molecule
- Prior art date
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
PHARMACEUTICALLY ACCEPTABLE COMPOSITIONS COMPRISING ANTIBODY MOLECULES SPECIFIC TO LAMININ-5 oc3 CHAIN DOMAINS G1 G2 AND USE THEREOF
FIELD OF THE INVENTION
The invention described herein primarily relates to pharmaceutically acceptable compositions comprising antibody molecules (e.g., antibodies and antibody "fragments") against laminin-5 and various methods of using such compositions and antibodies (e.g., in the treatment of cancer).
BACKGROUND OF THE INVENTION
The laminins are a family of matrix proteins composed of a variety of genetically dis- tinct alpha, beta, and gamma chains. Laminin-5, which recently has been referred to as Laminin-332 ("Ln332") (see Aumailley et al., Matrix Biol. 2005 Aug;24(5):326-32), for example, is composed of α3β3γ2 chains. The β3γ2 chains are exclusively found in laminin-5, whereas α3 chain is also present in laminin-6 and laminin-7 (although studies suggest in a different variant, often designated α3B). Laminin-5 (abbreviated "Ln5" or "Ln5" and also increasingly known as laminin-332,
Ln332, and the like) is a normal constituent of the basal membrane to which epithelial origin adhere. In cancer, the expression of laminin-5 is turned on suggesting laminin-5 assists and support in the tumor development and/or tumor progression. Expression of laminin-5 is strongly up-regulated in several types of solid tumors especially at the invasive front. Previously published research and literature has demonstrated and suggested that certain regions of Ln5 may provide suitable targets for antibodies having anti-cancer properties. WO 2005/056598, WO 2005/040219, and WO 2005/052003, for example, indicate that antibodies directed to the gamma-2 (γ2) chain of Ln5, particularly domain 3 thereof (γ2D3), may be useful anti-cancer agents. Previously published literature also suggests that the alpha3 (α3) chain of Ln5 may similarly be a useful target for anti-cancer antibodies (see, e.g., WO 2004/075835, US 20040009171 , and US 20030091569 providing general suggestion but without working examples or specific disclosures). Blocking tumor establishment in vivo was however described in Dajee et al. Nature 2003 vol. 421 p. 639 . The α3 chain of Ln5, which is known by several synonyms (e.g., epiligrin p200) has been well characterized in several species, including man (see, e.g., US Patent 6,120,991 ; Ryan et al., J Biol Chem. 1994 Sep 9;269(36):22779- 87; Galliano et al., J Biol Chem. 1995 Sep 15;270(37):21820-6). The carboxyl-terminally located globular (G) subdomains (the G1 to G5 domains - sometimes collectively referred to as the "G" domain) of the laminin-5 α3 chain are considered responsible for α3 integrin binding, by which cells bind to laminin-5. A number of in- tegrin pairs can bind laminin-5, including integrin α3β1 , α6β4, and α6β1 . The α6β1 integrin pair is more promiscuous binding many laminin molecules with essentially same affinity whereas the two first mentioned integrin pairs have strong preference for laminin-5. As such, and due to other functions exhibited by G1 -G5, the G domains are considered relevant to a number of cellular functions associated with Ln5, and G1 -G5 likely contains multiple cell binding sites with different mechanisms and different functions (Kariya et al., J. Cell Bio- chem., 88(3), 506-20 (2003)).
The G4-G5 subdomains in the Ln5 alpha3 chain is proteolytically cleaved in the production of mature α3(160 kDa)/Ln5, and appears to facilitate deposition of precursor α3 (190 kDa)/Ln5, and is likely important to hemidesmosome formation (Sigle et al., J Cell ScL, 1 17(pt19), 4481 -94 (2004); Baudoin et al., J Invest Dermatol., 125(5), 883-8 (2005)). The G3 subdomain has been reported to be very important in respect of cell adhesion and migration (Kariya et al., 2003, supra; Hirosaki et al., J Biol Chem 275: 22495-22502 (2000); US 20050220760). G2, G4, and G5 reportedly play roles in promoting cell adhesion (Kariya et al. (2003), supra) (G1 and G3 also are expected to play roles in promoting adhesion). Reports differ on the role of G4/G5 in cell migration and motility. WO 2005/073254 describes antibodies against the G4/G5 domains of Ln5α3 that reportedly exhibit anti-cancer properties. US 20020076736 suggests that antibodies directed to the intersection of the G3 and G4 domains may be useful in controlling cell migration. WO 2000/26342 suggests the use of various specific antibodies against Ln5α3 globular domains, generally (specifically discussed therein are antibodies ("Abs") BM165, CM6, 5C5, EM1 1 , and P3H9-2) for blocking cell proliferation. However, little is known about the characteristics of these or other previously reported anti-α3 antibodies. Antibody 5C5 is an anti-rat Ln5 antibody (see, Johnathan Jones Invention Abstract, "Laminin-5 and Hemidesmosomes," see http://ttp. northwestern. edu/abstracts/viewabs.php?id=18&cat=15), and apparently binds to the "long rod," rather than G domains, of α3 (see, e.g., Baker and Jones, Biol. Bull., 194, 400-401 (1998)). Antibody CM6 appears to be specific for a portion of processed (160 kD) α3, presumably, in an integrin-binding site, and may be capable of blocking cell adhesion and proliferation, but does not induce apoptosis (see, e.g., Baker et al, J.Cell Sci, 109, 2509-2520 (1996); Gonzales et al, MBC, 10, 259-270 (1999)). Antibody P3H9-2 is currently commercially available from Chemicon (Temecula, CA - USA), also ap- pears to target an integrin-binding site in α3, and reportedly also blocks adhesion and prolif- eration (see, e.g., Gonzales et al, MBC, 10, 259-270 (1999); Kim et al., J. Immunol., 165, 192-201 (2000); and Wayner et al., J. Cell Biol., 121 , 1 141 (1993)). Antibody EM1 1 , recognizes both intact (190 kDa) and processed (160 kDa) α3, but little else appears to be reported about the antibody (see, e.g., Ghosh et al., J. Biol. Chem., Vol. 275, Issue 31 , 23869- 23876 (2000)). Dajee et al. (2003) Nature vol.421 page 639, describe an anti-tumor effect of blocking laminin-5 by use of the monoclonal antibody (mAb) BM165. The BM165 antibody recognizes the α3 chain of laminin-5 (in the G1 -G3 domain region) and appears capable of blocking adhesion, migration, and proliferation of Ln5-associated cells (see also, e.g., Rous- selle et al., J. Cell Biol, 1 14, 567-576 (1991 )). Gonzales et al, MBC, 10, 259-270 (1999) and Goldfinger et al., J. Cell ScL, 1 12,
2615-2629 (1999) describe an anti-G2 antibody ("RG13") that apparently blocks migration and proliferation. Another anti-α3 antibody ("10B5") does not appear to have a characterized binding site and is not associated with any reports of inhibition of adhesion, migration, and/or proliferation (see Jones Invention Disclosure, supra and Goldfinger et al, J. Cell Biol., 141 , 255-265 (1998)). Another anti-α3 antibody (12C4) binds G5 (Goldfinger et al., J. Cell ScL, 1 12, 2615-2629 (1999)). Frank and Carter, J. Cell Sci, 1 17, 1351 -1363 (2004), describe several anti-α3 Abs (C2-9, which may bind an integrin binding site and appears to block adhesion; C2-5, which is non-inhibitory; and D2-1 , which binds G4-G5) (see also Xia et al, JCB, 132, 727-740 (1996)). Another anti-α3 Ab, P3E4, also is available from Chemicon, but ap- pears to be non-inhibitory (Wayner et al., J. Cell Biol. 121 :1 141 (1993)).
There remains a need for alternative and improved antibody molecules targeted to Ln5α3G1/G2, and particularly antibodies suitable for use as anti-cancer therapeutics. The invention described herein provides, inter alia, anti-α3G1/G2 antibody molecules having such properties, as well as other useful anti-G1/G2 antibody molecules, methods for producing such anti-G1/G2 antibodies, new and useful methods of using these and other anti-Ln5 antibodies, and other related methods and compositions.
SUMMARY OF THE INVENTION
The invention described herein provides novel antibody molecules that specifically bind ("are directed to" or "are directed against") the G1 and/or G2 domains ("G1/G2" or when these subdomains are presented together "G1 G2" or "G1 -G2") of the alpha3 ("α3") chain of human laminin-5 (hl_n5 or hl_n5) (which, as noted above, also may be referred to as laminin- 332 (Ln332 or Ln-332). This targeted area of the hl_n5 molecule may be referred to as "hl_n5α3G1/G2", "α3G1/G2," or simply "G1/G2," etc. Ln5α3 is known by a variety of synonyms including BM600-15OkDa, E170, epiligrin, Epiligrin 170 kDa subunit, A1 k, kalinin-165kDa, nicein-15OkDa, 200 kDa chain, and Nicein alpha subunit. The protein and gene (LAMA3) sequences for hl_n5α3 are widely reported in relevant databases as reflected in Table 1 :
Table 1 - Gene/Protein Sequence References for Ln5α3 UniProt Q16787, Q13680, Q6VU67
OMIM 226700, 245660
NCBI Gene 3909
NCBI RefSeq NP_937762, NP_000218
NCBI RefSeq NM_198129, NM_000227
NCBI UniGene 3909
NCBI Accession CAA59325, AAQ72571
An amino acid sequence for human Ln5α3 is provided under GenBank Accession No. AAA59483 (definitions of the G subdomains also are indicated), which is included herein as SEQ ID NO:1 1. Additional relevant sequences for α3 proteins can be found under Gen- Bank Accession Numbers AAH93406, NP_937762, NP_000218, and Q16787.
Unless otherwise stated, or clearly indicated by context, all references to laminin-5 herein are in respect of human laminin-5 (references to subunits of Ln5 should be similarly construed). Recombinant human Ln5 (rhLnδ or simply rl_n5) is described in, e.g., US 6703363. To the extent there are differences that impact any aspect of the invention, references to α3 herein should be construed as referring to the α3 chain of Ln5 (i.e., Ln332) (α3A) rather than that of Ln-6 or Ln-7 (α3B). However, it may be possible that various aspects of the invention are relevant to both forms of the laminin α3 chain.
In one exemplary aspect, the invention provides an antibody molecule that com- petes with monoclonal antibody (mAb) 7B2 (described further herein) more than mAb BM165 (BM165 is known in the public domain and is described in the Background hereto) for binding to hl_n5α3G1 G2. In particular aspects, the invention provides such antibodies, wherein the antibody binds to α3G2 (either solely or jointly with G1 ).
In another exemplary aspect, the invention provides an isolated antibody molecule that specifically binds Ln5α3G2 and that comprises either (a) CDR-L1 , CDR-L2, and CDR-L3 of mAb 7B2 (SEQ ID NOs:1 -3, respectively) or (b) CDR-H1 , CDR-H2, and CDR-H3 of mAb 7B2 (SEQ ID NOs:4-6, respectively).
In more particular exemplary aspects, the invention provides antibody molecules directed to G1/G2 (and typically some of the other above-described properties), wherein the molecules are capable of: (a) reducing hl_n5-associated cancer cell adhesion; (b) reducing migration of Ln5-associated cancer cells; and/or (c) inhibiting growth of Ln5-associated tumors. The term "inhibition" in this respect refers to any detectable decrease in the rate of growth, spread of growth, and/or amount of growth, etc., of such tumors; such that "inhibition" may be partial or total (in the latter case meaning that no growth of Ln5-associated tumors is observed in the treated area for a relevant period).
In yet another illustrative aspect, the invention provides an isolated antibody molecule that specifically binds an antigenic determinant region located in residues 1064-1077 of hl_n5α3 (SEQ ID NO:1 1 ). In a more particular illustrative aspect, the invention provides such an antibody molecule, wherein the molecule further binds to at least a portion of Ln5α3 defined by residues 989-1008 and/or 1082-1090 thereof.
In another exemplary aspect, the invention provides pharmaceutically acceptable compositions that comprise antibody molecules directed against domains G1 and/or G2 of hl_n5α3 and optionally characterized by any or all of the above-described properties. In yet another exemplary aspect, the invention provides a method of treating cancer comprising administering to a mammalian host such a composition. In another particular aspect, the invention provides a method of reducing cancer progression in a human patient suffering from or at substantial risk of developing an Ln5-associated cancer.
The Description of the Invention and other portions of this document provide further description of these aspects and disclose additional features and aspects of the invention.
DESCRIPTION OF THE DRAWINGS
Figure 1 - Flow cytometry (FACS) analysis for cell surface binding of mAbs BM165 and 7B2.
Figure 2 - Graph showing results of human tumor growth inhibition studies with mAbs BM165 and 7B2 in a mouse model.
Figure 3 - Graph showing results of another human tumor growth inhibition studies with mAbs BM165 and 7B2 in a mouse model.
Figure 4 - Graph showing results of tumor growth inhibition studies with mAb 7B2 and Erbitux in a mouse model. DESCRIPTION OF THE INVENTION
The invention provided here relates to novel antibody molecules that are capable of binding to Ln5 G1/G2 (i.e., G1 and/or G2), related compositions, and method of preparing and using such antibody molecules and compositions. In one exemplary aspect, the invention provides an isolated antibody molecule (e.g., a full-length antibody, an antibody fragment, or a derivative of either thereof) that competes with mAb 7B2 for binding to domain G2 of the α3 chain of hl_n5.
The term "antibody molecule" is used to refer to any molecule that is, or comprises, a G1/G2-specific (typically G2-specific) immunoglobulin (whether naturally occurring or not) or a G1/G2-specific immunoglobulin "fragment." Typically, an antibody molecule, in the context of the inventive methods and compositions described herein, refers to a full-length immunoglobulin ("antibody").
Immunoglobulins are structurally related proteins secreted by mammalian (e.g., human) B lymphocyte-derived plasma cells in response to the appearance of an antigen. The basic unit of each antibody is a monomer. An antibody molecule can be monomeric, dimeric, trimeric, tetrameric, pentameric, etc. The antibody monomer is a "Y"-shaped molecule consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter-connected by disulfide bonds.
The structure of immunoglobulins has been well characterized. See, e.g., FUNDA- MENTAL IMMUNOLOGY (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region typically is comprised of three domains, CH1 , CH2, and CH3. Each light chain typically is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant re- gion. The light chain constant region typically is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability (or hypervariable regions, which can be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). In full length, naturally produced antibod- ies, each VH and VL typically is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4 (which also may be referred to as FR L1 , CDR L1 , etc. or loop L1 , L2, L3 in the light chain variable domain and loop H1 , H2, and H3 in the heavy chain domain in the case of hypervariable loop regions (see, e.g., Chothia and Lesk J. MoI. Biol. 196:901 -917 (1987)). Typically, the numbering of amino acid residues in this region is performed by the method described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991 ) (phrases such as "variable domain residue numbering as in Kabat" and "according to Kabat" herein refer to this numbering system for heavy chain variable domains or light chain variable domains). Using this numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of CDR H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.
The carboxy-terminal portion of each antibody chain typically defines a constant region primarily responsible for effector function. Human light chains typically are classified as kappa and lambda light chains. Heavy chains typically are classified as mu, delta, gamma, alpha, or epsilon, and typically define an antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Unless otherwise stated or clearly contradicted by context, an antibody in the context of this invention can generally possess any suitable isotype (including, e.g., an isotype that is switched from its original isotype). Immunoglobulin G ("IgG") is the predominant immunoglobulin of internal components such as blood, cerebrospinal fluid and peritoneal fluid (fluid present in the abdominal cavity).
IgG is the only class of immunoglobulin that crosses the placenta, conferring the mother's immunity on the fetus. IgG makes up 80% of the total immunoglobulins. It is the smallest immunoglobulin, with a molecular weight of 150,000 Daltons. Thus it can readily diffuse out of the body's circulation into the tissues. All currently approved antibody drugs comprise IgG or IgG-derived molecules. As such, an immunoglobulin the context of this invention is typically an IgG molecule.
In some species, the immunoglobulin classes are further differentiated according to subclasses, adding another layer of complexity to antibody structure. In humans, for example, IgG antibodies comprise four IgG subclasses -- IgGI , lgG2, lgG3, and lgG4. Each subclass corresponds to a different heavy chain isotype, designated g1 (IgGI ), g2 (lgG2), g3 (lgG3), g4 (lgG4), a1 (IgAI ) or a2 (lgA2).
Antibody molecules can be selected based on their ability to provide or not provide complement fixation and/or complement dependent cytotoxicity (CDC). There are a number of isotypes of antibodies that are capable of complement fixation and CDC, including, without limitation, the following: murine IgM, murine lgG2a, murine lgG2b, murine lgG3, human IgM, human IgGI , and human lgG3. Those isotypes that are not capable of complement fixation/CDC include, without limitation, human lgG2 and human lgG4. lsotype determination and other methods for modifying the complement fixation and CDC functional characteristics of antibodies are known in the art.
The production of antibody molecules, by various means, is generally well understood. US Patent 6331415 (Cabilly et al.), for example, describes a method for the recombinant production of immunoglobulin where the heavy and light chains are expressed simultaneously from a single vector or from two separate vectors in a single cell. Wibbenmeyer et al., (1999, Biochim Biophys Acta 1430(2):191 -202) and Lee and Kwak (2003, J. Biotechnology 101 :189-198) describe the production of monoclonal antibodies from separately produced heavy and light chains, using plasmids expressed in separate cultures of E. coli. Various other techniques relevant to the production of antibodies are provided in, e.g., Har- low, et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).
In mammals (and certain other chordates), the reaction between antibodies and an antigen (which is usually associated with an infectious agent) leads to elimination of the antigen and its source. This reaction is highly specific, that is, a particular antibody usually reacts with only one type of antigen. The antibody molecules do not destroy the infectious agent directly, but, rather, "tag" the agent for destruction by other components of the immune system. In mammals such as humans, the tag is constituted by the CH2-CH3 part of the antibody, commonly referred to as the Fc domain.
The phrase "full length antibody" can be used to refer to any immunoglobulin molecule comprising at least most of the Fc domain and other domains commonly found in a naturally occurring immunoglobulin monomer. This phrase is used herein to emphasize that a particular antibody molecule is not an antibody fragment. As noted elsewhere, the terms antibody and immunoglobulin generally (unless contradicted explicitly or clearly by context) can be construed herein as referring to either full-length antibodies or immunoglobulin fragments. An "immunoglobulin fragment" or "antibody fragment" is any molecule that consists of or comprises a functional (i.e., G1/G2-specific) portion of an immunoglobulin. Antibody "fragments," may be produced by any suitable technique (e.g., enzymatic cleavage, peptide synthesis, and recombinant protein production) (i.e., the term "fragment" in no way limits the means by which such molecules are produced). Generally, any suitable antibody fragment can be used as a surrogate for a "full-length antibody" in the inventive compositions, meth- ods, and uses described herein, and visa versa, unless otherwise stated or clearly contradicted by context. Nonetheless, although having similar binding properties as full-length antibodies, the various types of antibody fragments described herein, collectively and each independently, may appropriately be considered unique features of the invention, exhibiting different biological and/or physiochemical properties than antibodies.
A suitable antibody fragment in the context of the inventive methods and compositions described herein may be, e.g.,: (i) a Fab fragment, a monovalent fragment consisting essentially of the VL, VH, CL and CH I domains; (ii) F(ab)2 and F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CH1 domains; (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists essentially of a VH domain; and (vi) one or more isolated CDRs or a functional paratope. Additional exemplary antibody fragments include Fab1 fragments, dsFv molecules, scFv molecules (and other single chain anti- body molecules), scFv dimers, minibodies, diabodies, F(ab') fragments, and the like. In one exemplary aspect, the invention provides an antibody fragment comprising a first polypeptide chain that comprises any of the heavy chain CDRs described herein and a second polypeptide chain that comprises any of the light chain CDRs described herein, wherein the two polypeptide chains are covalently linked by one or more interchain disulfide bonds. In a more particular aspect, the invention provides a two-chain antibody fragment having such features wherein the antibody fragment is selected from Fab, Fab1, Fab'-SH, Fv, and/or F(ab')2 fragments. Other exemplary antibody "fragments" include "kappa bodies" (see, e.g., Ill et al., Protein Eng 10: 949-57 (1997)) and "janusins." Previously published literature provides ample description of these and other antibody fragments and methods for the produc- tion thereof (see, e.g., WO2005040219). Other types of antibody fragment molecules and related antibody molecules are described in, e.g., US Patent Applications 20050238646, 20020161201 ,
The antibody molecules of the invention are characterized by, among other things, an ability to specifically bind to Ln5α3 G1/G2 (i.e., the G1 and/or G2 subdomains of the hu- man Ln332 α3 chain). The term "specific" herein refers to the preferential binding of an antibody molecule for G1/G2, or a specific portion thereof, over other portions of α3. Typically, an antibody molecule specific for α3 also selectively binds to α3 over other biological molecules in the context of a relevant physiological environment in which α3 or a molecule comprising α3 (e.g., Ln5) is found (e.g., the basal lamina, a population Ln5-associated tumor cells, the invasive front of an Ln5-associated tumor, etc.). However, it also may be possible that an antibody molecule of the invention, specific for G1/G2, may cross-react with the γ2 or β3 chain of Ln5 and/or other biological molecules that are presented in similar contexts as α3 or α3-associated peptides (e.g., Ln5).
The relative specificity of an antibody molecule for a particular target (e.g., G1 and/or G2) can be relatively determined by competition assays as described herein (see also, e.g., WO2005040219; US Patent 5,660,827; Saunal and Regenmortel, (1995) J. Immunol. Methods 183: 33-41 ; Harlow, et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988); Colligan et al., eds., CURRENT PROTOCOLS IN IMMUNOLOGY, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993); and Muller, Meth. Enzymol. 92:589-601 (1983)).
The specificity of an antibody molecule for G1/G2 (or a specific portion thereof) is conferred by the presence of a unique epitope (at least in the context of α3) located in G1/G2 that the antibody directly targets. An epitope is the area or region on an antigen to which a specifically-binding peptide (such as an antibody) binds. An antibody can also be character- ized by its specificity for an antigenic determinant region ("ADR" or "antigenic determinant"). An ADR refers to any portion of a peptide that comprises one or more epitopes or functional portions thereof (e.g., an immunodominant component of an epitope). An ADR may also comprise one or more amino acid residues not directly involved in antibody molecule binding, such as amino acid residues which are effectively blocked by the antibody molecule upon binding (in other words, the amino acid residue is within the "footprint" of the specific antigen binding peptide, as might be determined through HxMS (described further elsewhere herein)). Thus, an antigenic determinant in the context of this invention generally includes any peptide or peptide-derivative determinant capable of being specifically bound by an antibody molecule. An antibody molecule useful in the compositions and methods of the invention will have sufficient specificity for G1 and/or G2 to be able to readily bind α3 and remain bound for a significant amount of time (or "relevant period") under suitable conditions. In one aspect, a "significant amount of time" (or "relevant period") is any period that is sufficient to detect binding of the antibody molecule to G1/G2 in standard diagnostic methods (e.g., an ELISA). In another aspect, a "relevant period" is a period that is sufficient to induce, promote, or enhance a biological effect that is attendant binding of G1/G2 by the antibody molecule (e.g., an inhibition of Ln5-associated cell migration, a reduction in tumor progression, etc.). In the context of the inventive methods provided herein, a relevant period typically refers to a period of at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 4 hours, at least about 8 hours, or longer such as about 1 -12 hours, about 1 -24 hours, about 1 - 36 hours, about 1 -48 hours, about 1 -72 hours, etc., in which an antibody molecule is bound to G1/G2.
To exhibit sufficient binding to G1/G2, an antibody molecule used in the methods or compositions of the invention will exhibit a suitable level of affinity or avidity for G1/G2. As such, affinity and avidity provide another useful way of characterizing useful antibody molecules in the context of the compositions and methods of the present invention. "Affinity" refers to the strength of binding of an antibody molecule to an epitope or antigenic determinant. Typically, affinity is measured in terms of a dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag] where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant Ka is defined by 1/Kd. Suitable methods for determining binding peptide specificity and affinity by competitive inhibition, equilibrium dialysis, and the like can be found in, e.g., Harlow, et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988); Colligan et al., eds., CURRENT PROTOCOLS IN IMMUNOLOGY, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983). Generally, an antibody molecule will have affinity for G1/G2 in the range of about 104 to about 1010 M- 1 (e.g., about 107 to about 109 M'1). The term immunoreact herein typically refers to binding of an antibody molecule to G1/G2 (or a peptide comprising G1/G2, such as α3 or Ln5) with a dissociation constant (Kd) that is about 10'4 M or less. Affinity can be determined by any of the methods described elsewhere herein or their known equivalents in the art. An example of one method that can be used to determine affinity is provided in Scatchard analysis of Munson & Pollard, Anal. Biochem. 107:220 (1980). Binding affinity also may be determined by equilibrium methods (e.g. enzyme-linked immunoabsorbent assay (ELISA) or radioimmunoassay (RIA)) or kinet- ics analysis (e.g., BIACORE™ analysis).
It has been experimentally determined that mAb 7B2 has an affinity for Ln5 in the NM range (in one experiment an affinity of 1.67E-09 M was measured, in another experiment an affinity of 2.19E-09 M was measured). Similar affinities are expected to be exhibited by antibody molecules derived from mAb 7B2. Thus, in a particular aspect, the invention pro- vides antibodies having some or all of the above-described characteristics and that also may be characterized as having an affinity for Ln5 in the range of about 1 .5 x 10'9 M to about 2.5 x 10'9 M (e.g., about 2-3 x 10'9 M, such as about 2 to about 2.5 x 10'9 M).
Typically, the disassociation constant for antibody molecules used in the methods of the invention (or comprised in the compositions of the invention) is less than about 100 nM, less than about 50 nM, less than about 10 nM, about 5 nM or less, about 1 nM or less, about 0.5 nM or less, about 0.1 nM or less, about 0.01 nM or less, or even about 0.001 nM or less, in respect of G 1/G2.
"Avidity" refers to the overall strength of the total interactions between a binding protein and antigen. "Affinity," in contrast, is the strength of the total noncovalent interactions between a single antigen-binding site on an antibody or other binding peptide and a single epitope or antigenic determinant. "Avidity" typically is governed by three major factors: the intrinsic affinity of the binding protein for the epitope(s) or antigenic determinant(s) to which it binds, the valence of the antibody or binding protein and antigen (e.g., a multivalent antibody polymer will typically exhibit higher levels of avidity for an antigen than a bivalent antibody and a bivalent antibody can will have a higher avidity for an antigen than a univalent antibody, especially where there are repeated epitopes in the antigen), and/or the geometric arrangement of the interacting components. By generating polymeric molecules or antibodies with higher valences, even antibody monomers with relatively lower levels of affinity may be made that have sufficiently levels of avidity to be useful in the context of the inventive meth- ods and compositions described herein.
As described above, in one aspect the invention provides antibody molecules characterized by, among other things, the ability to compete with monoclonal antibody (mAb) 7B2 ("7B2") for binding of G2 (preferably in the context of Ln5). 7B2 is a mouse IgGI anti-G2 antibody, which the inventors isolated and characterized. The mature heavy variable chain of 7B2 is set forth as SEQ ID NO:7 and is encoded by SEQ ID NO:8. The mature light variable chain of 7B2 is set forth as SEQ ID NO:9 and is encoded by SEQ ID NO:10. The CDRs of 7B2 are set forth as SEQ ID NOs:1 -6 (CDR-H1 , H2, H3, L1 , L2, and L3, respectively).
Competition herein means a greater relative inhibition than about 30% as determined by competition ELISA analysis (exemplified in the Experimental Data section, below). It can be desirable to set a higher threshold of relative inhibition as a criteria/determinant of what is a suitable level of competition in a particular context (e.g., where the competition analysis is used to select or screen for new antibodies designed with the intended function of blocking the binding of another peptide or molecule to Ln5 (e.g., for blocking interaction of Ln5 with an Ln5-binding integrin, matrix metalloproteinase, heparin, or naturally occurring anti-Ln5 antibody)). Thus, for example, it is possible to set criteria for "competitiveness" wherein at least about 40% relative inhibition is detected; at least about 25% relative inhibition is detected; or at least about 45% relative inhibition is detected before an antibody is considered sufficiently competitive. In, for example, cases where epitopes belonging to competing antibodies are closely located in an antigen, competition can be marked by greater than about 50% relative inhibition (e.g., at least about 55% inhibition, at least about 60% inhibition, at least about 65% inhibition, at least about 70% inhibition, at least about 80% inhibition, or a higher level or range of relative inhibition (such as about 50-95% inhibition)). In another particular aspect, the invention provides an antibody molecule that competes with mAb 7B2 more than BM165 for binding to domains G1 -G2 of the α3 chain of hu- man laminin-5 (hl_n5) (hl_n5α3G1 -G2).
As previously noted, 7B2 is a monoclonal antibody. Typically, an antibody molecule in the context of the present invention is a monoclonal antibody (mAb) or a fragment thereof. In the context of this invention, a "monoclonal antibody" refers to a composition comprising a homogeneous antibody population having a uniform structure and specificity. Typically, a monoclonal antibody is an antibody obtained from a population of substantially homogeneous antibodies; i.e., the individual antibodies comprised in the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies commonly are highly specific, typically directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody (pAb) preparations that typi- cally include different antibodies directed against different antigenic determinants, each monoclonal antibody typically is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method unless specifically stated. For example, monoclonal antibodies to be used in accordance with various aspects of the present invention may be made by the hybridoma method, e.g., as first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., US Patent 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352:624-628 (1991 ) and Marks et al., J. MoI. Biol. 222:581 -597 (1991 ).
Monoclonal antibodies herein specifically include "chimeric" antibodies. The term "chimeric antibody" refers to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies (typically of antibodies of different species). Chimeric antibodies include monovalent, divalent, and polyvalent antibod- ies. A monovalent chimeric antibody typically is a dimer (HL) formed by a chimeric H chain associated through disulfide bridges with a chimeric L chain. A divalent chimeric antibody typically is a tetramer (H2L2) formed by two HL dimers associated through at least one disulfide bridge. A polyvalent chimeric antibody can also be produced, for example, by employing a CH region that aggregates (e.g., from an IgM H chain, or μ chain). Typically, a chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as frag- ments of such antibodies, so long as the fragments exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81 :6851 - 6855 (1984)). The production of chimeric antibodies is well understood in the art (see, e.g., WO2005040219).
In one aspect of the invention, antibody molecules that comprise sequences from mAb 7B2 in combination with sequences of other species (e.g., a chimeric antibody comprising G2 specific sequences from 7B2) are provided. Such antibody molecules may comprise any number of suitable modifications from 7B2 (e.g., modifications in the Fc region that enhance Fc functions). Any of these types of antibody molecules that comprise some portion of 7B2 (or another particular antibody) may be said to be "derived" from mAb 7B2 (the same principle may be applied to other "reference" antibodies).
An antibody molecule also may be a "humanized" monoclonal antibody (e.g., a humanized antibody comprising some or all of the CDRs of mAb 7B2). A "humanized" antibody is an antibody that is derived from a non-human species, in which certain amino acids in the framework and constant domains of the heavy and light chains have been mutated so as to avoid or abrogate an immune response in humans. Humanized forms of non-human (e.g., murine) antibodies are thus chimeric antibodies which contain at least (and usually only or little more than) minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervari- able region of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhu- man primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications typically are made to further refine antibody functionality and/or physiochemical properties. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. A humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immu- noglobulin. For further details regarding the characteristics and production of typical humanized antibodies, see, e.g., Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992), WO 92/02190, US Patent Application 20060073137, and US Patents 6,750,325, 6,632,927, 6,639,055, 6,548,640, 6,407,213, 6,180,370, 6,054,297, 5,929,212, 5,895,205, 5,886,152, 5,877,293, 5,869,619, 5,821 ,337, 5,821 ,123, 5,770,196, 5,777,085, 5,766,886, 5,714,350, 5,693,762, 5,693,761 , 5,530,101 , 5,585,089, and 5,225,539. In one aspect, the invention provides humanized antibodies that are "derived" from mAb 7B2.
Another more recent approach that may be applied to generate useful variants of 7B2 (or variants of other non-human, e.g., murine, anti-G1/G2 antibodies) for therapeutic applications in humans (e.g., comprising the CDRs of mAb 7B2), is to generate antibody molecules having increased human "string content" by the methods described in, e.g., US Patent Application 20060008883. This method is the subject of antibody molecules produced by Xencor, Inc. Antibody molecules may also be antibody variants that also or alternatively are modified by other deimmunization methods (i.e., methods designed to reduce the immuno- genicity of the antibody molecule in a human patient). Such antibodies included veneered antibodies (see, e.g., US Patent 6,797,492 and US Patent Applications 20020034765 and 20040253645) and antibodies that have been modified by T-cell epitope analysis and re- moval (see, e.g., US Patent Application 20030153043 and US Patent 5,712,120) (see also, Gonzales et al., Tumour Biol. 2005 Jan-Feb;26(1 ):31 -43; Lantto et al., J Biol Chem. 2002 Nov 22;277(47):45108-14); Chowdury et al., Methods. 2005 May;36(1 ):1 1 -24; and Holliger et al., Nat Biotechnol. 2005 Sep;23(9):1 126-36).
Antibody molecules also can include variations in other portions of the antibody structure. For example, the Fc region of the antibody molecule can be modified so as to increase Fc functions (see, e.g., US Patent Applications 20040132101 , 20050054832, 20050249723, 20060134105, 20060074225, and 20060024298 - a method also used in various molecules produced by Xencor, Inc.). Variations in other regions also may improve antibody functionality, without impairing G1/G2 specificity (see, e.g., US Patent Applications 20050244403 and 200401 10226). US Patent Application 2003077613 provides other methods for generating variant antibody molecules with useful properties. Other approaches to improving Fc function (e.g., by modifying antibody molecule glycosylation) that may also or alternatively be applied to generate antibody variant molecules useful in the context of the methods and compositions provided by the invention are described in, e.g., US Patent Appli- cation Nos. 2005272916, 20050272128, 20050123546, 20050079605, 20040241817, and 20040072290. These methods have been applied to produce novel antibody molecules by companies such as Biowa and Glycart.
In another exemplary aspect, the invention provides an isolated antibody molecule that specifically binds Ln5α3G2 and that comprises either (a) CDR-L1 , CDR-L2, and/or CDR- L3 of mAb 7B2 (SEQ ID NOs:1 -3, respectively) or (b) CDR-H1 , CDR-H2, and/or CDR-H3 of mAb 7B2 (SEQ ID NOs:4-6, respectively). In one aspect, the invention provides an antibody molecule that comprises at least a complete set of heavy chain CDRs from 7B2 or light chain CDRs from 7B2. In a particular aspect, the invention provides an antibody molecule that comprises CDR-H1 , CDR-H2, and CDR-H3 of mAb 7B2 and at least some of CDR-L1 , CDR- L2, and CDR-L3 of mAb 7B2. In a more particular aspect, the invention provides an antibody molecule wherein the antibody molecule comprises CDR-L1 , CDR-L2, and CDR-L3 of mAb 7B2 and CDR-H1 , CDR-H2, and CDR-H3 of mAb 7B2.
In another aspect, the invention provides antibody molecules that comprise a VH domain having at least about 80% identity (e.g., at least 85%, 90%, 95%, 97%, or more iden- tity) to the VH domain of mAb 7B2 (SEQ ID NO:7). In another aspect, the invention provides an antibody molecule that also or alternatively comprises a VL domain having at least about 80% identity (e.g., at least 85%, 90%, 95%, 97%, or more identity) to the VL domain of mAb 7B2 (SEQ ID NO:8). In a particular aspect, the invention provides antibody molecules that comprise essentially all of the VH and/or VL domains of mAb 7B2. Functional antibody molecules comprising only a portion of the complement of
CDRs of a mammalian antibody have been successfully generated by a variety of known methods. "Single domain" antibody fragments and other antibody molecules comprising only a portion of mAb 7B2, are another feature of the invention. In other words, these types of antibody molecules may be derived from mAb 7B2 using known techniques to provide addi- tional antibody molecules useful in the compositions and methods of the invention.
So-called "domain antibodies" (dAbs) for example, being developed by Domantis, correspond to the VH regions of either the heavy or light chain of an antibody (the heavy and light chain variable regions for mAb 7B2 are provided as SEQ ID NOs:7 and 8) (such molecules and methods for producing such molecules are described in, e.g., US Patents 6,248,516, 6,696,245, 6,291 ,158; 6,582,915; 6,593,081 ; and 6,172,197; US Patent Application 200401 10941 ; EP 1433846, EP0368684, and EP0616640; International patent Applications WO05/035572, WO04/101790, WO04/081026, WO04/058821 , WO04/003019 and WO03/002609; and Holt et al., Trends Biotechnol. 2003 Nov;21 (1 1 ):484-90; Muyldermans et al., Trends Biochem Sci. 2001 Apr;26(4):230-5; Muyldermans, J Biotechnol. 2001 Jun;74(4):277-302; Reichmann et al., J Immunol Methods. 1999 Dec 10;231 (1 -2):25-38; and Dick, BMJ. 1990 Apr 14;300(6730):659-60. Camelids (camels, llamas, and alpacas) and sharks produce antibodies that display single chain high-affinity VH-domain-only antibodies, rather than an Fv module, and that therefore bind to their target antigens using just three CDR loops. These VH domains are typically referred to as "VHHs." A relatively enlarged hypervariable region allows such molecules to exhibit a broad antigen-binding repertoire, despite the lack of combinatorial diversity associated with regular antibody variable regions. Human V-like domain proteins ("camelized" human VH antibodies) have been successfully adapted as a scaffold for display and selection using large CDR loops that can penetrate clefts in the target antigen. Such humanized V-like domains can be a suitable framework for targeting "clefts" and/or "canyons" in target proteins. Bispecific VHH antibodies have been generated by tethering two single-domain antibody fragments with the structural upper hinge of a typical antibody and other VHH antibodies have been successfully targeted to cancer- associated targets. Such VHH, camelized, and VHH-associated bispecific antibodies are described in, e.g., Conrath et al., J Biol Chem. 2001 Mar 9;276(10):7346-50; and Cortez- Retamozo et al., lnt J Cancer. 2002 Mar 20;98(3):456-62 (see also EP1558645, WO2004041865, and US2005214857, for descriptions of related molecule types).
Typically, antibody molecules of the invention may be characterized as "isolated molecules." An isolated molecule is a molecule that is the predominant species in the composition wherein it is found with respect to the class of molecules to which it belongs (i.e., it makes up at least about 50% of the type of molecule in the composition and typically will make up at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more of the species of molecule, e.g., peptide, in the composition). Commonly, a composition of an antibody molecule will exhibit 98%, 98%, or 99% homogeneity for antibody molecule in the context of all present peptide species in the composition or at least with respect to substantially active peptide species in the context of proposed use. For example, a peptide stabilizer/buffer such as an albumin may be intentionally included in a final pharmaceutical formulation, without impeding the activity of an antibody molecule. In the context of a composition comprising one or more pharmaceutically acceptable carriers, an antibody molecule can be present in relatively small amounts in terms of numbers of total molecular species in the composition (e.g., in the case of a composition comprising a large amount of a pharmaceutically acceptable carrier, stabilizer, and/or preservative). For example, additional peptides, such as BSA, can be included in such a composition with a previously purified antibody molecule. However, provided that such additional constituents of the composition are acceptable for the intended application of the antibody molecule, such a composition may still be described as comprising an "isolated" antibody molecule, unless otherwise indicated or clearly contradicted by context.
An isolated molecule also or alternatively typically refers to a molecule that is not associated with significant amounts (e.g., more than about 1%, more than about 2%, more than about 3%, or more than about 5%) of extraneous and undesirable biological molecules, e.g., undesirable molecules contained within a cell, cell culture, chemical media, or animal in which the antibody molecule is produced.
An isolated molecule also or alternatively can refer to a molecule that has passed through such a stage of purity due to non-natural (human) intervention (whether automatic, manual, or both) for a significant amount of time (e.g., at least about 10 minutes, at least about 20 minutes, at least an hour, or longer).
In one facet of the invention, the antibody molecule of the compositions or methods described herein is characterized by, La., the ability to reduce Ln5-associated cancer cell adhesion. Thus, in one aspect, the invention provides a method of reducing cancer cell adhe- sion comprising contacting a population of such cells with an antibody molecule of the invention under conditions suitable for reducing the adhesion of such cells (which may be in vitro or in vivo - e.g., in a mammalian subject, such as a human patient). Cell adhesion assays for Ln5 associated cells are described in, e.g., US Patent Application 20040120959. Moreover, the adhesion properties of laminin-5 have been demonstrated and evaluated in several cell attachment studies (see, e.g., Carter et al., (1991 ) Cell 65: 599-610; Rousselle et al., (1991 ) J. Cell Biol. 1 14: 567:576; Sonnenberg et al., (1991 ) J. Cell Biol. 1 13: 907-917; Niessen, et al., (1994) Exp. Cell. Res. 21 1 : 360-367; Rousselle et al. (1994) J. Cell Biol. 125:205214); Rousselle et al., J Biol Chem. 1995 Jun 9;270(23):13766-70; Kim et al., Exp Cell Res. 2005 Mar 10 ;304(1 ):317-27; Kariya et al., J Cell Biochem. 2003 Feb 15;88(3):506-20; Hirosaki et al., J Biol Chem. 2000 JuI 21 ;275(29):22495-502; and SaIo et al., Matrix Biol. 1999
Apr;18(2):197-210) Additional assays studying the adhesion effects of anti-α3G2 mAb 7B2 are included in the Experimental Methods section of this document.
In another aspect, an antibody molecule used in the compositions or methods of the invention also or alternatively typically can be characterized by having the ability to reduce migration of Ln5-associated cancer cells, and related physiological phenomenon (e.g., Ln5- associated tumor budding and/or invasiveness). Thus, in one aspect, the invention provides a method for reducing migration of Ln5-associated cancer cells in a medium (e.g., a cell culture, a mammalian host (e.g., a human patient), etc.) that comprises contacting such cells with an amount of an antibody molecule of the invention under conditions suitable for reduc- ing migration of the Ln5-associated cells. Methods suitable for assessing migration and related physiological phenomenon include Boyden and Transwell chamber assays (see, e.g., US 20020052307; Hujanen and Terranova (1985) Cancer Res. 45: 3517-3521 ; and Pelletier, A.J., Kunicki, T. and Quaranta, V. (1996), J. Biol. Chem. 271 :364); matrigel migration assays (see, e.g., Zhang et al., Onco- gene. 2004 Apr 15;23(17):3080-8 and Knutson et al., Molecular Biology of the Cell, 7: 383- 396, 1996); radiographic assays (such as barium radiographic invasiveness assays); positron emission tomography assessments; magnetic resonance imaging (MRI) techniques (e.g., measurement of tumor diameter and/or volume); biopsy. The characterization of invasive cells is well known in the art. A discussion of invasive cell characteristics and related principles can be found in, e.g., King RJB (1996) CANCER BIOLOGY (Addison Wesley Longman Ltd., Harlow Essex) and Liotta and Stetler-Stevenson (1991 ) Cancer Res 51 :5054s- 5059s. Studies involving migration and Ln5 are reported in, e.g., Fukushima et al., lnt J Cancer. 1998 Mar 30;76(1 ):63-72; Calaluce et al., Neoplasia. 2004 Sep-Oct;6(5):468-79; Frank et al., J Cell Sci. 2004 Mar 15;1 17(Pt 8):1351 -63; Pirila et al., Biochem Biophys Res Commun. 2003 Apr 18;303:1012-7; Tsuji et al., Clin Exp Metastasis. 2002;19(2):127-34; Decline et al., J CeII Sci. 2001 Feb;1 14(Pt 4):81 1 -23; Plopper et al., Breast Cancer Res Treat. 1998 Sep;51 (1 ):57-69; and Masaki et al., Anticancer Res. 2003 Sep-Oct;23(5b):41 13-9.
A detailed exemplary protocol for a Transwell assay of cell migration is provided in this paragraph for purposes of illustrating a diagnostic tool that, like other assays described herein, can be used in the context of assessing the suitability of an antibody molecule. Transwell plates with pore size of 12μm can be obtained from Costart (Cambridge, MA, USA). The lower side of the membrane can be coated with about 2.5 μg of EHS type IV collagen overnight (o/n) at room temperature (RT). Suitable cells, for example HSC-3 cells, are removed from cell culture, and incubated with or without test antibody molecules, such as anti- G2 mAbs (typically about 25, 50, or 100 μg/ml of the test mAb, normal mouse IgG, unspecific mAb, and/or nothing (control) is used in such assays), for a suitable time (e.g., about 30 minutes) at a suitable temperature (typically about 37 0C). The cells can then be transferred to the upper part of the chamber with antibodies and allowed to migrate for a sufficient period of time (typically about 6 hours), with or without addition of FCS and optionally in the presence of a low concentration (e.g., 0.1 %) of BSA. A sufficient amount of FN can be used as a chemoattractant in the lower part of the chamber (e.g., 2.5 μg/ml). Cells from the upper side of the membrane can be removed and cells that have migrated through the membrane can be stained and the number of cells calculated under a microscope by field (typically the view is divided into a circular area of 10 fields diameter). Such assays, using for example, HSC-3 cells, may be used to demonstrate, e.g., the impact of antibody molecules on cell motility. In one aspect, the invention provides antibody molecules that are capable of reducing cancer cell adhesion to a point that apoptosis of such cells is enhanced.
In a particular aspect, the invention provides an antibody molecule that is capable of inhibiting migration of SCC-25 cells with an IC50 of about 0.02 μg/ml. In another aspect, the invention provides antibody molecules that also or alternatively are capable of inhibiting migration of A431 cells with an IC50 of about 0.05 μg/ml. In still another aspect, the invention provides antibody molecules that also or alternatively are capable of inhibiting migration of MDA-MB-231 cells with an IC50 of about 0.04 μg/ml. In another aspect, the invention provides antibody molecules that are capable of inhibiting migration of cancer cells with a po- tency that is equal or greater than BM165 cells (e.g., in SCC-25, A431 and/or MDA-MB-231 cells, particularly in A431 and MDA-MB-231 cells). An exemplary method for assessing inhibition of cancer migration by antibody molecules is provided in the Experimental Methods section of this document.
In yet another aspect, the invention provides antibody molecules that also or alterna- tively may be characterized by their ability to inhibit growth of Ln5-associated tumors. In a particular aspect, the invention provides antibody molecules having the ability to inhibit such growth in vivo. "Inhibiting tumor growth" generally means causing a reduction in the amount of tumor growth that would otherwise occur (in the absence of treatment) and/or substantially complete cessation of detectable tumor growth, and includes decreases in tumor size and/or decrease in the rate of tumor growth. Methods for assessing inhibition of tumor growth are well known in the art. Publications examining the effect of Ln5 and Ln5-related molecules on tumor growth include, e.g., Miyazaki et al., Cancer Sci. 2006 Feb;97(2):91 -8. The Experimental Methods section of this document also includes an exemplary method for assessing tumor growth. In another aspect, the invention provides antibody molecules that also or alternatively may be characterized by being capable of reducing tumor volume (either total or average). In a more particular aspect, the invention provides antibody molecules that are at least as effective as mAb BM165 in reducing tumor volume. In one aspect, an effective amount of an antibody molecule of the invention is capable of achieving an at least 2-fold reduction in tumor volume in a mammal as compared to a substantially similar untreated mammal. In a more particular aspect, an effective amount of an antibody molecule of the invention is capable of achieving an at least 3-fold reduction in tumor volume as compared to a substantially similar untreated mammal. In another aspect, the invention provides antibody molecules that are also or alternatively able to be characterized based on their ability to detectably bind colon epithelium cells and/or blood vessel cells, but to not bind smooth muscle cells.
Antibody molecules of the invention are intended to exclude those anti-α3G1/G2 an- tibodies previously known, such as BM165. Thus, in certain aspects, an antibody molecule of the invention may be characterized as having any of the above-described characteristics, or combinations thereof, with the proviso that the antibody molecule is not mAb CM6, C2-9, 10B5, RG13, 12C4, CM6, D2-1 , P3E4, C2-5, P3H9-2, C2-9, 10B5, RG13, 5C5, EM1 1 , and/or BM165 or an antibody molecule derived therefrom. Another feature of at least some of the antibody molecules of the invention is the ability to bind Ln5 at the surface of cancer cells. Thus, antibody molecules of the invention can be characterized by exhibiting this feature alternatively or in addition to any of the above- described characteristics. The ability to bind to proteins at the surface of the cell can be determined by any suitable method (e.g., flow cytometry). Application of a flow cytometry method to determine antibody molecule binding at the cell surface is described in the Experimental Methods section of this document.
Another feature of at least some of the antibody molecules of the invention is the ability to cross-react with the mouse basal membrane and human basal membrane.
Additional examples of antibody variant molecules that may generated using stan- dard techniques from antibodies provided by the invention (e.g., 7B2) and that can be useful in the compositions and methods of the invention include so-called superantibodies (e.g., dimerizing superantibodies with enhanced effector potency). Such molecules are described in, e.g., Zhao et al., Drug Discov Today. 2005 Sep 15;10(18):1231 -6; Kohler et al., Immunol Today. 1998 May;19(5):221 -7; Kohler, Appl Biochem Biotechnol. 2000 Jan-Mar;83(1 -3):1 -9; discussion 10-2, 145-53 (see also, International Patent Application WO 02/097041 ). Still another type of variant antibody molecule that can be engineered from antibodies provided herein is an immunoadhesin (see, e.g., US Patent Application 20040033561 ). Other exemplary antibody variants (fusion proteins) are described in, e.g., US Patent 6,589,527 (see also, generally, Presta et al., J Allergy Clin Immunol. 2005 Oct;1 16(4):731 -6). Still another type of exemplary antibody variant molecule that can be generated from antibodies provided by the invention is so-called Abzymes, which are antibody-like molecules that have the ability to catalyze chemical reactions. Abzymes are briefly described in, e.g., WO2005040219 and relevant references cited therein.
An additional aspect of the invention is embodied in "derivatives" of any of the above-described antibody molecules. A "derivative" in this context can be considered any protein, e.g., an antibody molecule, in which one or more of the amino acid residues of the protein have been chemically modified (e.g., by alkylation, acylation, ester formation, amide formation, or other similar type of modification) or covalently associated with one or more heterologous substituents (e.g., a lipophilic substituent, a PEG moiety, a peptide side chain linked by a suitable organic moiety linker, etc.). The second type of derivative can separately be described as a "conjugate."
In general, antibody molecules described herein can be modified by inclusion of any suitable number of such modified amino acids and/or associations with such conjugated substituents. Suitability in this context general is determined by the ability to at least substan- tially retain α3 G1/G2 selectivity and/or specificity associated with the non-derivatized parent antibody molecule (and therefore also implies retention of a suitable level of affinity and/or avidity). The inclusion of one or more modified amino acids may be advantageous in, for example, (a) increasing polypeptide serum half-life, (b) reducing polypeptide antigenicity, or (c) increasing polypeptide storage stability. Amino acid (s) are modified, for example, co- translationally or post-translationally during recombinant production (e.g., N-linked glycosyla- tion at N-X-S/T motifs during expression in mammalian cells) or modified by synthetic means. Non-limiting examples of modified amino acids that may make up part of a derivative include glycosylated amino acids, sulfated amino acids, prenlyated (e.g., farnesylated, geranylgeranylated) amino acids, acetylated amino acids, acylated amino acids, PEGylated amino acids (see, e.g., US Patent Application 200601 10382 and Chapman et al., Adv Drug DeNv Rev. 2002 Jun 17;54(4):531 -45, Chapman et al, Nat Biotechnol. 1999 Aug;17(8)780-3, Chowhurdy et al., Methods. 2005 May;36(1 ):1 1 -24, and Roberts et al., Adv Drug DeNv Rev. 2002 Jun 17;54(4):459-76), biotinylated amino acids, carboxylated amino acids, phosphory- lated amino acids, and the like. References adequate to guide one of skill in the modification of amino acids are replete throughout the literature. Exemplary protocols are found in
Walker (1998) PROTEIN PROTOCOLS ON CD-ROM Humana Press (Towata, NJ). Typically, a modified amino acid in a derivative is a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, a lipid moiety-conjugated amino acid residue, or an organic derivatizing agent-conjugated residue. Additionally, antibodies and antibody fragments can be chemically modified by cova- lent conjugation to a polymer to increase their circulating half-life, for example. Exemplary polymers and methods to attach such polymers to peptides are illustrated in, e.g., US Patents 4,766,106; 4,179,337; 4,495,285; and 4,609,546. Additional illustrative polymers include polyoxyethylated polyols and polyethylene glycol (PEG) moieties (e.g., an antibody molecule can be conjugated to a PEG with a molecular weight of between about 1 ,000 and about 40,000, such as between about 2000 and about 20,000, e.g., about 3,000-12,000).
In another aspect, the invention provides an antibody molecule having some or all of the above-described properties that also is conjugated to a second molecule that is selected from a radionuclide, an enzyme, an enzyme substrate, a cofactor, a fluorescent marker, a chemiluminescent marker, a peptide tag, a magnetic particle, a toxin, or other drug. Another exemplary feature of the invention is an antibody molecule that is conjugated to one or more antibody fragments, nucleic acids (oligonucleotides), nucleases, hormones, immunomodula- tors, chelators, boron compounds, photoactive agents, dyes, and the like. These and other suitable agents can be coupled either directly or indirectly to antibody molecules of the invention. One example of indirect coupling of a second agent is coupling by a spacer moiety. These spacers, in turn, can be either insoluble or soluble (see, e.g., Diener, et al., Science, 231 :148, 1986) and can be selected to enable drug release from the antibody molecule at a target site and/or under particular conditions. Additional examples of therapeutic agents that can be coupled to antibody molecules include lectins and fluorescent peptides.
In another aspect, the invention provides crosslinked antibody molecule derivatives. For example, an antibody molecule derivative can be produced by crosslinking two or more antibodies, at least one of which is specific/selective for α3 G1 G2 (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuc- cinimidyl suberate). Such linkers are available from Pierce Chemical Co., Rockford, III. Antibody molecules also can be conjugated with any suitable type of chemical group, such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These and other suitable conjugated groups may be used to improve the biological characteristics of the antibody molecule, e.g., to increase serum half-life, solubility, and/or tissue binding.
Antibody molecule derivatives can be produced by chemically conjugating a radioisotope, protein, or other agent/moiety/compound to, for example, (a) the N-terminal side or C-terminal side of the antibody molecule or subunit thereof (e.g., an anti-α3 G1 G2 antibody H chain, L chain, or anti-α3 G1 G2 specific/selective fragment thereof), (b) an appropriate substituent group or side chain or (c) a sugar chain associated with the antibody molecule (see, e.g., ANTIBODY ENGINEERING HANDBOOK, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)). Derivatives also can be generated by conjugation at internal residues or sugars, where appropriate and available. In one aspect, a derivatizing agent is a low molecular weight compound. Examples include anticancer agents, such as alkylating agents (e.g., nitrogen mustard, cyclophosphamide); metabolic antagonists (e.g., 5-fluorouracil, methotrexate); plant alkaloids (e.g., vincristine, vinblastine, vindesine); hormone drugs (e.g., tamoxifen, dexamethasone), and the like (see, e.g., CLINICAL ONCOLOGY, edited by Japanese Society of Clinical Oncology, published by Cancer and Chemotherapy (1996)).
In one aspect, antibody molecule derivatives comprising one or more radiolabeled amino acids are provided. A radiolabeled antibody molecule may, for example, be used for both diagnostic and therapeutic purposes (conjugation to separate radiolabeled molecules is another possible feature). Nonlimiting examples of labels for conjugation to peptides and/or incorporation in amino acid residues include, but are not limited to 3H, 14C, 15N, 35S, 90Y, 99Tc, and 1251, 131 I, and 186Re. Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (see, e.g., Junghans et al. in CANCER CHEMOTHERAPY AND BIOTHERAPY 655-686 (2d edition, Chafner and Longo, eds., Lippincott Raven (1996)) and US Patents 4,681 ,581 , 4,735,210, 5,101 ,827, 5,102,990 (U.S. Re. Pat. No. 35,500), 5,648,471 , and 5,697,902. For example, a radioisotope can be conjugated by a chloramine T method. Antibody molecules having one or more of the above-described features that also are linked to cytotoxins are another useful feature of the invention. Cytotoxic drugs which can be conjugated to antibody molecules, such as anti-α3 G1 G2 antibodies, and used for in vivo therapy include, but are not limited to, daunorubicin, mercaptopurine, adriamycin, doxorubicin, methotrexate, and Mitomycin C. Cytotoxic drugs can interfere with critical cellular processes including DNA, RNA, and protein synthesis. For a description of these classes of drugs which are well known in the art, and their mechanisms of action, see Goodman, et al., GOODMAN AND GiLMAN's THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 8th Ed., Mac- millan Publishing Co., 1990. Additional techniques relevant to the preparation of antibody immunotoxins are provided in, e.g., Vitetta, Immunol. Today 14:252 (1993) and U.S. Pat. No. 5,194,594. Cytotoxic proteins, such as pseudomonas exotoxin, also can be conjugated or linked to an antibody molecule (several examples of such proteins are described elsewhere herein with reference to antibody molecule fusion proteins). Additional examples of toxic molecules that can be conjugated to antibody molecules include diphtheria toxin (e.g., diphtheria A chain and active fragments thereof) and related molecules (e.g., hybrid molecules) (see, e.g., US Patent 4,675,382), ricin toxin (e.g., a deglycosylated ricin A chain toxin) (see, e.g., Vitetta et al., Science 238, 1098 (1987) and US Patent 4,643,895), cholera toxin, a Shiga-like toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxins. Conju- gates of the monoclonal antibody and such cytotoxic moieties can be made using a variety of bifunctional protein coupling agents. Examples of such reagents include SPDP, IT, bifunc- tional derivatives of imidoesters such a dimethyl adipimidate HCI, active esters such as disuccinimidyl suberate, aldehydes such as glutaraldehyde, bis-azido compounds such as bis (p-azidobenzoyl) hexanediamine, bis-diazonium derivatives such as bis-(p- diazoniumbenzoyl)-ethylenediamine, diisocyanates such as tolylene 2,6-diisocyanate, and bis-active fluorine compounds such as 1 ,5-difluoro-2,4-dinitrobenzene. The lysing portion of a toxin typically may be readily joined to the Fab fragment/portion of an antibody or antibody fragment. Other suitable conjugated molecules include ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, diphtherin toxin, and Pseudomo- nas endotoxin. See, for example, Pastan et al., Cell 47:641 (1986), and Goldenberg, Calif.--A Cancer Journal for Clinicians 44:43 (1994). Additional toxins suitable for use in the present invention are known (see, e.g., US Patent 6,077,499).
In one aspect, the invention provides an antibody molecule that is conjugated to a mixed toxin. A mixed toxin molecule is a molecule derived from two different (typically poly- peptide) toxins. Generally, peptide toxins comprise one or more domains responsible for generalized eukaryotic cell binding, at least one enzymatically active domain, and at least one translocation domain. The binding and translocation domains are required for cell recognition and toxin entry respectively. Naturally-occurring proteins which are known to have a translocation domain include diphtheria toxin, Pseudomonas exotoxin A, and possibly other peptide toxins. The translocation domains of diphtheria toxin and Pseudomonas exotoxin A are well characterized (see, e.g., Hoch et al., Proc. Natl. Acad. Sci. USA 82:1692, 1985; Co- lombatti et al., J. Biol. Chem. 261 :3030, 1986; and Deleers et al., FEBS Lett. 160:82, 1983), and the existence and location of such a domain in other molecules may be determined by methods such as those employed by Hwang et al. (Cell 48:129, 1987); and Gray et al. (Proc. Natl. Acad. Sci. USA 81 :2645, 1984). In view of these techniques, a useful mixed toxin hybrid molecule can be formed, for example, by fusing the enzymatically active A subunit of E. coli Shiga-like toxin (Calderwood et al., Proc. Natl. Acad. Sci. USA 84:4364, 1987) to the translocation domain (amino acid residues 202 through 460) of diphtheria toxin, and to a molecule targeting a particular cell type, as described in US Patent 5,906,820. The targeting portion of the three-part hybrid can cause the molecule to attach specifically to the targeted cells, and the diphtheria toxin translocation portion can act to insert the enzymatically active A subunit of the Shiga-like toxin into a targeted cell. The enzymatically active portion of Shiga-like toxin, like diphtheria toxin, acts on the protein synthesis machinery of the cell to prevent protein synthesis, thus killing the targeted cell. Additionally useful conjugate substituents include anti-cancer retinoids, taxane conjugates (see, e.g., Jaime et al., Anticancer Res. 2001 Mar-Apr;21 (2A):1 1 19-28), cisplatin conjugates, thapsigargin conjugates, linoleic acid conjugates, calicheamicin conjugates (see, e.g., Damle et al., Curr Opin Pharmacol. 2003 Aug;3(4):386-90), doxorubicin conjugates, geldanamycin conjugates, and the like, also may be useful in promoting the treatment of cancer (see, generally, Trail et al., Cancer Immunol Immunother. 2003 May;52(5):328-37). In another aspect, an antibody molecule is conjugated to a tumor targeting domain peptide or molecule. In one example, an antibody molecule is conjugated to a tumor targeting factor VII sequence.
In another aspect, the invention provides antibody molecules conjugated to or oth- erwise stably associated with one or more detection-facilitating agents (i.e., detection agents, tags, or labeling moieties). Useful detection agents with which an antibody molecule may be derivatized include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1 -napthalenesulfonyl chloride, lanthanide phosphors, and the like. Additional examples of suitable fluorescent labels include a 125Eu label, an isothiocy- anate label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an o- phthaldehyde label, a fluorescamine label, etc. Examples of chemiluminescent labels include luminal labels, isoluminal labels, aromatic acridinium ester labels, imidazole labels, ac- ridinium salt labels, oxalate ester labels, a luciferin labels, luciferase labels, aequorin labels, etc. An antibody molecule also can be labeled with enzymes or enzyme substrates that are useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. An antibody molecule also can be labeled with biotin, and accordingly detected through indirect measurement of avidin or streptavidin binding. An antibody molecule may also be labeled with a predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.). Additional examples of enzyme conjugate candidates include malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triose phosphate isomerase, asparaginase, glucose oxidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. Additional exemplary labeling moieties generally include, but are not limited to spin- labeled molecules and other labeling moieties of diagnostic value (e.g., molecules that act as contrast agents in MRI diagnosis).
In another aspect, the invention provides an antibody molecule that is conjugated to an immunomodulator, such as an immunomodulating cytokine, stem cell growth factor, lym- photoxin (e.g., a TNF such as TNFα), or a hematopoietic factor. Examples of such molecules that may be useful as conjugates include IL-1 , IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21 , colony stimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF)), interferons (e.g., IFNα, IFNβ, and IFNγ), the stem cell growth factor designated "S1 factor," erythropoietin, and throm- bopoietin, active fragments thereof, derivatives thereof, variants thereof, or a combination of any thereof. Examples of immunomodulating agents (e.g., various cytokines, T cell activity modulators, NK cell activity modulators, etc.) are described elsewhere herein.
In another exemplary aspect, an antibody molecule or related compound/molecule (such as an antibody molecule-encoding nucleic acid, an antibody molecule related antigenic peptide, etc.) is conjugated to or otherwise associated with a functional nucleic acid molecule. Functional nucleic acids include antisense molecules, interfering nucleic acid molecules (e.g., siRNA molecules), aptamers, ribozymes, triplex forming molecules, and external guide sequences. Methods of producing such derivatized antibody molecules are described in, e.g., WO2005040219.
A "variant" of an antibody is an antibody molecule comprising an antibody derived amino acid sequence that differs from the sequence of the "parent" antibody sequence (nearest related (by sequence identity) reference sequence, which usually is the sequence of a mammalian-produced antibody) by one or more suitable amino acid residue substitutions, deletions, insertions, or terminal sequence additions in at least the CDRs or other VH and/or VL sequences (provided that at least a substantial amount of the epitope binding characteristics of the parent antibody are retained, if not improved upon, by such changes). Antibody molecules having such modifications in respect of parent antibodies, e.g., antibody molecules comprising one or more changes in the CDRs of mAb 7B2, are another aspect of the invention and may be suitably used in the inventive methods described herein.
In the design, construction, and/or evaluation of CDR "variants" attention can be paid to the fact that CDR regions can vary to enable a better binding to the epitope. Antibody CDRs typically operate by building a "pocket," or other paratope structure, into which the epitope fits. If the epitope is not fitting tightly, the antibody may not offer the best affinity. How- ever, as with epitopes, there often are a few key residues in a paratope structure that ac- count for most of this binding. Thus, CDR sequences can vary in length and composition significantly between antibodies for the same peptide. The skilled artisan will recognize that certain residues, such as tyrosine residues (e.g., in the context of CDR-H3 sequences), that are often significant contributors to such epitope binding, are typically desirably retained in the "construction" and "design" of a CDR variant.
A convenient way for generating substitution variants is affinity maturation using phage using methods known in the art. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis also can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively or addition- ally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues are likely suitable candidates for substitution.
Useful methods for rational design of CDR sequence variants are described in, e.g., International Patent Applications WO 91/09967 and WO 93/16184. Additional considerations in the production/selection of peptide variants (e.g., conservation of amino acid residue functional characteristics, conservation of amino acid residues based on hydropathic characteristics, and/or conservation of amino acid residues on the basis of weight/size, are described elsewhere herein). Typically, amino acid sequence variations, such as conservative substitution variations, desirably do not substantially change the structural characteristics of the par- ent sequence (e.g., a replacement amino acid should not tend to disrupt secondary structure that characterizes the function of the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in, e.g., Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); INTRODUCTION TO PROTEIN STRUCTURE (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991 )); and Thornton et at. Nature 354:105 (1991 ). Additional principles relevant to the design and construction of peptide variants are discussed in, e.g., Collinet et al., J Biol Chem 2000 Jun 9;275(23):17428-33.
Typically, advantageous sequence changes are those that (1 ) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity of the variant se- quence (typically desirably increasing affinity), and/or (4) confer or modify other physico- chemical or functional properties on the associated variant/analog peptide. In the context of CDR variants, particularly in the context of variant anti-G1/G2 antibodies, it is typically desired that residues required to support and/or orientate the CDR structural loop structure(s) are retained; that residues which fall within about 10 angstroms of a CDR structural loop (but optionally only residues in this area that also possess a water solvent accessible surface of about 5 angstroms2 or greater) are unmodified or modified only by conservative amino acid residue substitutions; and/or that the sequence is subject to only a limited number of insertions and/or deletions (if any), such that CDR structural loop-like structures are retained in the variant (a description of related techniques and relevant principles is provided in, e.g., Schiweck et al., J MoI Biol. 1997 May 23;268(5):934-51 ; Morea, Biophys Chem. 1997
Oct;68(1 -3):9-16; Shirai et al., FEBS Lett. 1996 Dec 9;399(1 -2):1 -8; Shirai et al., FEBS Lett. 1999 JuI 16;455(1 -2):188-97; Reckzo et al., Protein Eng. 1995 Apr;8(4):389-95; and Eigen- brot et al., J MoI Biol. 1993 Feb 20;229(4):969-95).
Amino acid sequence variations can result in an altered glycosylation pattern in the variant antibody with respect to a parent antibody. By "altering", it is meant deleting one or more carbohydrate moieties found in the parent antibody, and/or adding one or more glycosylation sites that are not present in the parent antibody. 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 and asparagine-X-threonine, where X is any amino acid except proline, are common 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 typically can create a potential glycosylation site. O-linked glycosylation refers to the attachment of sugars such as N-aceylgalactosamine, 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 can be 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).
From the foregoing it will be understood that the invention also provides for antibody molecules that comprise amino acid sequences derived from antibodies described herein, such as 7B2, that include modifications in the CDRs thereof. Typically, however, an antibody molecule derived from a particular reference antibody, such as 7B2, will retain most of the CDR sequence of the reference antibody. If changes are made to the CDRs, the changes are subject to analysis to ensure that important characteristics of the "parent" antibody (e.g., binding of G2 with a similar level of affinity) are retained. The phrase "potential amino acid interactions" can be used to refer to contacts or energetically favorable interactions between one or more amino acid residues present in an antigen and one or more amino acid residues which do not exist in a parent antibody but can be introduced therein so as to increase the amino acid contacts between the antigen and an antibody variant comprising those introduced amino acid residue(s). Desirably, antibody variants having modifications in the CDRs or other regions that effect target binding are associated with increased potential amino acid interactions with G1/G2. Amino acid interactions of interest can be selected from hydrogen bonding interactions, van der Waals interactions, and/or ionic interactions.
Alanine scanning mutagenesis techniques, such as described by Cunningham and Wells (1989), Science 244:1081 -1085, can be used to identify suitable residues for substitution or deletion in generating variant VL, VH, or particular CDR sequences, although other suitable mutagenesis techniques also can be applied. Multiple amino acid substitutions also can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer, Science 241 :53-57 (1988) or Bowie and Sauer Proc. Natl. Acad. Sci. USA 86:2152-2156 (1989). Additional techniques that can be used to generate variant antibodies and antibody molecules modified in target binding sequences include the directed evolution and other variant generation techniques described in, e.g., US 20040009498; Marks et al., Methods MoI Biol. 2004;248:327-43 (2004); Azriel-Rosenfeld et al., J MoI Biol. 2004 Jan 2;335(1 ):177-92; Park et al., Biochem Biophys Res Commun. 2000 Aug 28;275(2):553-7; Kang et al., Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):1 1 120-3; Zahnd et al., J Biol Chem. 2004 Apr 30;279(18):18870-7; Xu et al., Chem Biol. 2002 Aug;9(8):933-42; Border et al., Proc Natl Acad Sci U S A. 2000 Sep 26;97(20):10701 -5; Crameri et al., Nat Med. 1996 Jan;2(1 ):100-2; and as more generally described in, e.g., International Patent Applications WO 2003048185 and WO2005040219.
In another aspect, the invention provides antibody molecules that are specific for G1/G2 as well as at least one "second" or "secondary" target. In other words, multispecific antibody molecules, having at least some of the above-described features, are another as- pect of the invention. In general, a multispecific antibody molecule used in the methods of the invention or incorporated of the compositions of the invention can have any suitable number of valencies and specificities. An anti-α3 G1 -G2 antibody molecule can be, e.g., a univalent antibody molecule. Antibody molecules with more than two valencies also can be prepared using known techniques and used in the context of the inventive methods de- scribed herein and incorporated into the compositions of this invention. For example, tris- pecific antibodies that are partially specific for α3 G1 -G2 can be prepared by methods known in the art (see, e.g., Tutt et al. J. Immunol. 147: 60 (1991 )).
In one exemplary aspect, the invention provides a bispecific antibody comprising at least one pair of VH sequence and VL sequence chains specific for an epitope comprised at least in part in α3 G1 -G2 and a second at least one pair of VH and VL sequence chains specific for a second (i.e., different) epitope. The VH and VL sequences in such a bispecific antibody can comprise complete VH and VL sequences corresponding to anti-α3 G1 -G2 antibody VH and VL region sequences, variant VH and/or VL sequences, and/or suitable portions of VH and/or VL regions, such as a combination of CDR sequences and other se- quences (e.g., framework sequences/residues) sufficient to provide binding to an epitope or epitopes of interest.
Bispecific antibodies can be produced by a variety of known methods including fusion of hybridomas or linking of Fab1 fragments (see, e.g., Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990) and Kostelny et al. J. Immunol. 148:1547-1553 (1992)). Traditionally, the recombinant production of bispecific antibodies is based on the co- expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (see, e.g., Milstein and Cuello, Nature, 305: 537 (1983)). Methods relevant to the production and purification of such molecules are also described in, e.g., WO 93/08829 and Traunecker et al., EMBO J., 10: 3655 (1991 ) (see also, Holliger and Winter 1993 Curr. Opin. Biotech. 4, 446-449; Poljak, R. J., et al. (1994) Structure 2:1 121 -1 123; and Cao et al. (1998), Bioconjugate Chem. 9, 635-644; US Patent Application 20030078385; Marvin and Zhu, Acta Pharmacologica Sincia, 26(6):649-658 (2005); and Kontermann, Acta Pharacol. Sin., 26:1 -9 (2005)).
"Full length" bi-specific antibodies (BsAb-IgG) (BsAbs comprising a functional anti- body Fc domain) also have previously been created, typically by chemical cross-linking of two different IgG molecules (Zhu et al 1994 Cancer Lett., 86, 127-134) or co-expressing two immunoglobulin G molecules ("IgGs") in hybrid hybridomas (Suresh et a/ 1986 Methods En- zymol 121 , 210-228). Brennan et al., Science, 229: 81 (1985), for example, describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments may then be reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab1 fragments generated can then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives can then be reconverted to the Fab'-thiol by reduction with mercap- toethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form a bispecific antibody.
According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences by recombinant or synthetic methods. The variable domain sequence is typically fused to an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. A first heavy-chain constant region (CH1 ), containing the site necessary for light chain binding, also typically is present in at least one of the fusion peptides. In a more specific example of this type of approach, a bispecific antibody is produced comprising 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. Such an asymmetric structure can facilitate the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations (such an approach is described in WO 94/04690). For further description of related methods for generating bispecific antibodies see e.g., Suresh et al., Methods in Enzymology, 121 :210 (1986). In yet another approach, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture so as to form a population of bispecific antibody molecules. Typically, such an interface comprises at least a part of the CH3 domain of an antibody constant region. Normally in such a method, one or more amino acid residues with smaller side chains from the interface of the first antibody molecule are replaced with amino acid residues with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain amino acid residue(s) are created on the interface of the second antibody molecule by replacing large amino acid side chain residues with smaller ones (e.g., alanine or threonine). This technique provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. Cross-linked or "heteroconjugate" antibodies are another type of bispecific antibody provided by the invention. Derivatives of such antibodies also can be advantageous for certain applications. For example, one of the antibodies in a heteroconjugate can be coupled to avidin and the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (see, e.g., U.S. Pat. No. 4,676,980). Heteroconju- gate antibodies may be made using any convenient cross-linking methods. Suitable peptide cross-linking agents and techniques are well known in the art, and examples of such agents and techniques are disclosed in, e.g., U.S. Pat. No. 4,676,980. Bispecific antibody "fragments," also can be generated - e.g., Fab'-SH fragments recovered from E. coli can be chemically coupled to form bispecific Abs. Shalaby et al., J. Exp. Med., 175: 217-225 (1992), for example, describe production of a humanized bispecific F(ab')2 molecule.
A wide variety of recombinant methods for producing multispecific antibody molecules are known. US Patent 6331415 (Cabilly et al.), describes a method for the recombinant production of immunoglobulin where the heavy and light chains are expressed simultaneously from a single vector or from two separate vectors in a single cell. Wibbenmeyer et al., (1999, Biochim Biophys Acta 1430(2):191 -202) and Lee and Kwak (2003, J. Biotechnol- ogy 101 :189-198) describe the production of monoclonal antibodies from separately produced heavy and light chains, using plasmids expressed in separate cultures of E. coli. Various other techniques for making and isolating bispecific antibody fragment molecules directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers (see, e.g., Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992)). The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) also has provided an alternative mechanism for making bispecific antibody fragment molecules (see also Alt et al., FEBS Letters, 454 (1990) 90-94 for a description of similar diabody-related techniques). Another strategy for making bispecific antibody fragment molecules by the use of single-chain Fv
(sFv) dimers has also been reported. See, e.g., Gruber et al., J. Immunol., 152:5368 (1994). In addition, bispecific antibodies may be formed as "Janusins" (Traunecker et al., EMBO J 10:3655-3659 (1991 ) and Traunecker et al., lnt J Cancer Suppl 7:51 -52 (1992)). Additional methods relevant to the production of multispecific antibody molecules are disclosed in, e.g., Fanger et al., Immunol. Methods 4:72-81 (1994).
Exemplary bispecific antibody and antibody-like molecules comprise (i) two antibodies one with a specificity to α3 G1 -G2 and another to a second target that are conjugated together, (ii) a single antibody that has one chain specific to α3 G1 -G2 and a second chain specific to a second molecule, and (iii) a single chain antibody that has specificity to α3 G1 - G2 and a second molecule. Typically, the second target/second molecule is a molecule other than α3, and usually other than Ln5. In one aspect, the second molecule is a cancer antigen/tumor-associated antigen such as carcinoembryonic antigen (CEA), prostate specific antigen (PSA), RAGE (renal antigen), α-fetoprotein, CAMEL (CTL-recognized antigen on melanoma), CT antigens (such as MAGE-B5, -B6, -C2, -C3, and D; Mage-12; CT10; NY- ESO-1 , SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin antigens (e.g., MUC1 , mucin- CA125, etc.), a cancer-associated ganglioside antigen, tyrosinase, gp75, C-myc, Marti , MelanA, MUM-1 , MUM-2, MUM-3, HLA-B7, or Ep-CAM. Additional cancer antigens that can be targeted by multispecific molecule are known (see, e.g., WO2005040219).
In another aspect, the invention provides a multispecific antibody that specifically binds a portion of α3 (typically a portion of G1 -G2) and at least one second molecule that is a cancer-associated integrin, such as α5β3 integrin.
In another aspect, the invention provides a multispecific antibody that specifically binds to a portion of α3 and at least one second molecule that is an angiogenic factor or other cancer-associated growth factor, such as a vascular endothelial growth factor (VEGF), a fibroblast growth factor (FGF), epidermal growth factor (EGF), epidermal growth factor re- ceptor (EGF-R), angiogenin, and receptors thereof, particularly receptors associated with cancer progression (e.g., one of the HER1 -HER4 receptors).
In a further facet, the invention provides a multispecific antibody that specifically binds to a portion of α3 and at least one second molecule that is a Ln5-associated modula- tion of cell migration, such as integrin α3β1 ; one or more other Ln5 associated integrins (e.g., alphaθbetai (α6β1 ), and alpha6beta4 (α6β4) integrins); type VII collagen; fibulin-2; IL-7, heat shock protein 27; BP180; syndecan-4; dystroglycan; nidogen-1 ; Lnδ/cancer-associated matrix metalloproteinases (e.g., MMP-1 , MMP-2, MMP-3, MMP-8, MMP-9, MMP-12, MMP-13, MMP-20, and MMP-14 (also known as MT1 -MMP) (see, e.g., Pirila et al., Biochem Biophys Res Commun. 2003 Apr 18;303(4):1012-7 with respect to examples of MMP processing of Ln5)); EWI (see, e.g., Stipp et al., J Cell Biol. 2003 Dec 8;163(5):1 167-77); tissue inhibitor of matrix metalloproteinase-1 (TIMP-1 ) and TIMP-2; BB94 (see, e.g., Giles et al., J Cell Sci. 2001 Aug;1 14(Pt 16):2967-76); tetraspanin D6.1A (see, e.g., Herlevsen et al., J Cell Sci. 2003 Nov 1 ;1 16(Pt 21 ):4373-90); CD151 (Winterwood NE et al. MoI. Biol. Cell 2006 Vol. 17 p 2707) and bone morphogenic protein-1 (BMP-1 - see, e.g., US Patent Application US 20020076736 with respect to methods involving BMP-1 and related molecules).
Other cancer progression-associated proteins discussed herein also or alternatively can be suitable second molecules targeted by multispecific antibody molecules (see, for example, the discussion of combination therapies provided elsewhere herein). Antibody molecule "fusion proteins" represent another exemplary feature of the invention. In a particular exemplary aspect, the invention relates to the use of such an antibody molecule in the preparation of medicaments for the treatment of conditions associated with oc3, such as Ln5-associated cancers. In another aspect, such a fusion protein can be used in various inventive methods described herein. Antibody molecule fusion proteins typically comprise (a) any suitable sequence or combination of sequences specific and/or selective for α3 (e.g., an anti-α3 G1 G2 antibody VH domain, VL domain, or particular CDRs thereof) and (b) at least one nonhomologous and typically substantially dissimilar amino acid sequence that imparts a detectable biological function and/or physiochemical characteristic to the fusion protein that cannot solely be at- tributed to the α3-specific/selective sequence(s) (e.g., binding of a non-α3-associated target, increased in vivo half-life, fluorescence, increased targeting to a particular type of cell, etc.). Such at least one substantially dissimilar sequence can be referred to as a "second sequence," "secondary sequence" or "fusion partner." A substantially dissimilar sequence typically has less than about 40% amino acid sequence identity to the α3-binding sequence(s), such as less than about 35%, less than about 30%, less than about 25%, or less than about 20% identity to the α3 G1 G2-specific/selective sequence(s).
The functional sequences of a fusion protein can be separated by one or more linkers, which typically can be characterized as flexible linker(s). Secondary sequence(s) can be derived from cytotoxic or apoptotic peptides (examples of peptides from which such sequences can be derived are described elsewhere herein). In this sense, similar to in the provision of various cytotoxic derivative molecules by the invention (discussed above), the invention provides a means for targeting a cytotoxic, apoptotic, or otherwise toxic payload to α3-associated tissues and cells. Secondary sequences also can confer diagnostic properties, such as fluorescence or enzymatic detection. Examples of such sequences include those derived from easily visualized enzymes, such as horseradish peroxidase. Enzyme derivatizing agents also may be suitable fusion partners in antibody molecule fusion proteins.
Antibody molecule fusion proteins also or alternatively can be characterized by comprising an epitope tag. An epitope tag is an amino acid sequence having enough residues to provide an epitope against which an antibody can be made, in the context of the antibody molecule, yet is short enough such that it does not substantially interfere with the activity (selectivity, specificity, affinity, and/or biological activity) of the antibody molecule (as compared to a "parent" antibody molecule lacking the epitope tag). An epitope tag desirably is sufficiently unique so that the anti-epitope tag antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least about 6 amino acid residues and usually between about 8-50 amino acid residues (e.g., about 9-30 residues). Examples of epitope tags include the flu HA tag polypeptide and its antibody 12CA5 (Field et al. (1988), MoI. Cell. Biol. 8:2159-2165); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al. (1985), MoI. Cell. Biol. 5(12):3610-3616); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al. (1990), Protein Engineering 3(6):547-553 (1990)). In certain embodiments, the epitope tag is a "salvage receptor binding epitope". As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgGi, IgG2, IgG3, or IgG4) that is respon- sible for increasing the in vivo serum half-life of the IgG molecule.
In another aspect, an antibody molecule fusion protein is provided wherein the fusion protein comprises a "fusion partner" sequence that corresponds to or consists essentially of a cytokine or biologically active fragment thereof (or a variant or derivative of either thereof), such as a cytokine or cytokine fragment that induces, promotes, and/or enhances immune cell activity (particularly anti-cancer immune cell activity) in vivo. Examples of such cytokines include interleukin 2 (IL-2), granulocyte macrophage colony-stimulating factor, interferon gammas (IFNγs), macrophage colony-stimulating factor, interleukin 12, and the like (additional immunomodulatory cytokines described elsewhere herein in the context of other aspects of the invention also or alternatively can be incorporated in such fusion proteins). A large number of cytokine variants and cytokine derivatives have been described in the art. Conjugate derivative antibody molecules comprising linked cytokine or active cytokine fragment peptides are another feature of the invention.
In another exemplary aspect, the invention provides an antibody molecule that comprises a suitable leucine zipper sequence fusion partner that can increase the affinity and/or production efficiency of the antibody molecule fusion protein as compared to a protein consisting or consisting essentially of the antibody molecule sequence(s). Potentially suitable leucine zipper sequences include the jun and fos leucine zippers taught by Kostelney et al. (1992), J. Immunol., 148: 1547-1553, and the GCN4 leucine zipper. Cytokine fusion proteins are further described in, e.g., Helguera et al., Clin Immunol. 2002 Dec;105(3):233-46 and Penichet et al., J Immunol Methods. 2001 Feb 1 ;248(1 -2):91 -101 .
Numerous methods for producing derivatives of proteins, including antibody molecules, are known in the art, which may be useful in, e.g., introduction of spacers into an antibody molecule fusion protein. Methods for coupling and site-specifically conjugating PEG to a Fab' fragment, for example, are described in Leong et al, Cytokine 16(3):106-1 19 (2001 ) and Delgado et al, Br. J. Cancer 73(2):175-182 (1996). PEG spacers typically have a MW of about 2000-4000. Such spacers can be used to conjugate derivatizing moieties or also to form fusion proteins by joining of different binding protein (typically antibody or antibody- derived, such as antibody fragment) portions. Relatively shorter spacers, for example short amino acid sequence spacers, such as a DSSP spacer, also similarly can be used to join an- tibody portions and/or derivatizing agents to antibody portions. Other linkers which also may be suitable are described herein and/or are known in the art (see, e.g., Kortt et al., Biomol Eng. 2001 Oct 15;18(3):95-108, regarding principles relevant to selection of linkers for single chain Fv antibody fragments). Antibody portions, such as Fab fragments or Fab-comprising antibody molecules also can be joined by Cys-Cys linkages, which can be facilitated by vari- ous known techniques. Joining of amino acid chains to linked moieties typically is accomplished by chemical crosslinking (such as by the affinity cross-linking methods described in US Patent 6,238,667). Pharmaceutically active and acceptable small molecules, radioactive compounds, and the like can be associated with fusion proteins in the form of chelates that attach to a molecule (e.g. biotin, avidin, streptavidin, etc.) that specifically binds an epitope tag in or attached to a fusion protein. Chelating groups are well known and include groups derived from ethylene diamine tetra-acetic acid (EDTA), diethylene triamine penta-acetic acid (DTPA), cyclohexyl 1 ,2-diamine tetra-acetic acid (CDTA), ethyleneglycol-O,O'-bis(- 2- aminoethyl)-N,N,N',N'-tetra-acetic acid (EGTA), N,N-bis(hydroxybenzyl)-e- thylenediamine- N,N'-diacetic acid (HBED), triethylene tetramine hexa-acetic acid (TTHA), 1 ,4,7,10- tetraazacyclododecane-N,N'-,N",N'"-tetr- a-acetic acid (DOTA) (see, e.g., US Patent
5,428,156 and Lewis et al. (1994) Bioconjugate Chem. 5: 565-576), hydroxyethyldiamine tri- acetic acid (HEDTA), 1 ,4,8,1 1 -tetra-azacyclotetradecane-N,N',N",N'"-tetra-acetic acid (TETA), substituted DTPA, substituted EDTA, and the like.
To inhibit cancer and reduce the number of preneoplastic cells by killing such cells, a toxin such as ricin, diphtheria toxin, anthrax toxins (see, e.g., Frankel et al., Curr Protein Pept Sci. 2002 Aug;3(4):399-407), eosinophil-derived neurotoxin, a tumor necrosis factor (e.g., TNFα), a mistletoe toxin (e.g., mistletoe lectin I A chain), abrin, saporin, Pseudomonas exotoxin, and the like, or a cytotoxic fragment thereof, or a combination of any thereof, can be incorporated in an antibody molecule fusion protein as a fusion partner (see generally, e.g., Frankel et al., Clinical Cancer Research Vol. 6, 326-334 (2000); Frankel, Clinical Cancer Research Vol. 8, 942-944 (2002); Brinkman et al., Expert Opin Biol Ther. 2001 Jul;1 (4):693-702; and Fitzgerald et al., Diagn. Ther. 7:447-62 (1992)). Apoptotic agents also can be suitable fusion partners, such as TNF-related apoptosis-inducing ligand (TRAIL/APO- 2L), PML, apoptin, and the like (see, e.g., Wajant, Apoptosis. 2002 Oct;7(5):449-59; see also generally, e.g., Thorbun et al., Apoptosis. 2004 Jan;9(1 ):19-25). In another aspect, a cytotoxic RNase peptide fusion partner can be used (e.g., a fusion partner based on onconase or ribonuclease A). Fusion partner sequences also can be derived from cytotoxic or apoptotic peptides (e.g., can be cytotoxic fragments of such sequences or cytotoxic variants of such sequences, etc.), examples of peptides from which such sequences can be derived are de- scribed elsewhere herein (e.g., with respect to antibody conjugates). In general, any peptide conjugate described elsewhere herein can be used as a fusion partner in an antibody fusion protein (and visa versa).
From the foregoing, it should be clear there are a large number of fusion partner sequences that can be advantageously included in an antibody molecule fusion protein. Fusion partner sequence can confer, for example, diagnostic properties, such as through facilitating fluorescence or enzymatic detection of the antibody molecule. Examples of such sequences include those derived from easily visualized enzymes, such as horseradish peroxidase, and fluorescent sequences, such as Green Fluorescent Protein (GFP) sequences. Antibody molecule "flourobodies" are, for example, another aspect of the invention. Fluorobodies are molecules made by grafting a functional set of CDRs (and typically associated resi- dues/sequences as framework therefore) onto a GFP sequence or other partner that emits a strong fluorescent signal.
In another exemplary aspect, the invention provides antibody molecule fusion proteins or conjugates comprising a cyclic or multi-cyclic (e.g., double cyclic) partner, such as a cyclic tumor homing peptide, e.g., the cyclic CNGRC peptide or double-cyclic
ACDCRGDCFC peptide (see, e.g., Ellersby et al., Nature Med., 5(9):1032-1038 (1999)). Similar cyclic homing peptides are known (see, e.g., Laakkonen, Nat Med. 2002 Jul;8(7):751 - 5). Antibody molecule fusion proteins can also comprise other homing/targeting domains, such as integrin-binding RGD domains and the like. In a further aspect, the invention provides G1/G2-specific antibody molecule adzymes. Adzymes, are proteins containing a binding domain and a separate enzyme active site which together act on a therapeutic target. The protein binding domain attaches to the disease-causing target, allowing the enzyme domain to abolish the function of the target. Antibody molecule fusion proteins can include any suitable number of any suitable types of linkers (between domains), such as a predominantly GIy and/or Ser flexible linker of about 5-20 amino acid residues, and may also or alternatively comprise a cleavable linker or cleavage site for proteinases, such as an enterokinase. Recombinant methods related to the insertion of linker sequences are well know (see, e.g., Sambrook et al., loc. cit., Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N. Y. (1989)). Additional suitable linkers can comprise oligomerization domains.
Methods of generating fusion proteins, which can be applied to the production of antibody fusion proteins and other fusion proteins provided by the invention are well known in the art. Such methods and related principles are described in, e.g., US Patents 5,457,035, 5,563,046; 5,668,225; 5,698,679; 5,763,733; 5,908,626; 5,969,109; 6,008,319; 6,1 17,656; 6,121 ,424; 6,132,992; 6,207,804; 6,224,870; and Borrebaeck et al., ANTIBODY ENGINEERING (2nd Ed., Oxford University Press 1995); WO93/10151 ; MOLECULAR CLONING (Cold Spring Harbor Press) (cited elsewhere herein); Ashkenazi et al. (1991 ) PNAS 88, 10535; Byrn et al. (1990) Nature 344, 677; and Hollenbaugh et al. (1992) "Construction of Immunoglobulin Fusion Proteins," in Current Protocols in Immunology, Suppl. 4, pp. 10.19.1 to 10.19.1 1 . Addi- tional methods and principles relevant to Ab fusion proteins are described in ANTIBODY FUSION PROTEINS (Chamov and Ashkenazi, Eds.) (Wiley-ϋss, 1999).
In another aspect, the invention provides a nucleic acid comprising a sequence that codes for production of a recombinant antibody molecule having one or more of the above- described features. An antibody molecule-encoding nucleic acid can have any suitable characteristics and comprise any suitable features or combinations thereof. Thus, for exam- pie, an antibody molecule-encoding nucleic acid may be in the form of DNA, RNA, or a hybrid thereof, and may include nonnaturally-occurring bases, a modified backbone (e.g., a phosphothioate backbone that promotes stability of the nucleic acid), or both. The nucleic acid advantageously comprises features that promote desired expression, replication, and/or selection in target host cell(s). Examples of such features include an origin of replication component, a selection gene component, a promoter component, an enhancer element component, a polyadenylation sequence component, a termination component, and the like, numerous suitable examples of which are known.
In a further aspect, the invention provides a vector comprising an antibody molecule- encoding nucleic acid (e.g., a nucleic acid comprising an antibody molecule-encoding sequence). A vector refers to a delivery vehicle that promotes the expression of an antibody molecule-encoding nucleic acid, the production of an antibody molecule peptide, the trans- fection/transformation of target cells, the replication of the antibody molecule-encoding nucleic acid, promotes stability of the nucleic acid, promotes detection of the nucleic acid and/or transformed/transfected cells, or otherwise imparts advantageous biological and/or physio- chemical function to the antibody molecule-encoding nucleic acid. Unless otherwise stated, a vector in the context of this invention can be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (a nucleic acid sequence comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one exemplary aspect, an antibody molecule-encoding nucleic acid is comprised in a naked DNA or RNA vector, including, for example, a linear expression element (as described in, e.g., Sykes and Johnston (1997) Nat Biotech 17: 355-59), a compacted nucleic acid vector (as described in, e.g., US Patent 6,077, 835 and/or International Patent Application WO 00/70087), a plasmid vector such as pBR322, pUC 19/18, or pUC 1 18/1 19, a "midge" minimally-sized nucleic acid vector (as described in, e.g., Schakowski et al. (2001 ) MoI Ther 3: 793-800), or as a precipitated nucleic acid vector construct, such as a CaP04-precipitated construct (as described in, e.g., International Patent Application WO 00/46147, Benvenisty and Reshef (1986) PNAS 83: 9551 -55, Wigler et al. (1978), Cell 14:725, and Coraro and Pearson (1981 ) Somatic Cell Genetics 7:603). Such nucleic acid vectors and the usage thereof are well known in the art (see, e.g., US Patents 5,589,466 and 5,973,972).
In another aspect, the invention provides secondary antibodies that bind to antibody molecules of the invention (e.g., mAb 7B2). A secondary antibody refers to an antibody spe- cific for, and typically raised against, another antibody, such as anti-α3 G1 G2 antibody (al- though antibodies against other antibody molecules can be similarly used/incorporated where applicable). An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody (e.g., a G2 epitope). Such antibodies are another feature of the invention. In another aspect, the invention provides pharmaceutically useful and acceptable compositions for administration to a mammalian host, such as a human patient, that comprise an amount (dosage) of one or more antibody molecules having features such as those described above. In general, an antibody molecule can be combined with one or more pharmaceutically acceptable carriers (diluents, excipients, and the like) and/or adjuvants ap- propriate for one or more intended routes of administration to provide compositions that are pharmaceutically acceptable. Pharmaceutically acceptable compositions comprising a therapeutic does of an antibody molecule of the invention also may be referred to as "pharmaceutical compositions". Acceptability of a composition and its components is generally made in terms of toxicity, adverse side effects, immunogenicity, etc., by standard methods. Pharmaceutically acceptable carriers generally include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible with an antibody molecule. Examples of pharmaceutically acceptable carriers include water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations of any thereof. In many cases, it can be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in such a composition. Pharmaceutically acceptable substances such as wetting agents or minor amounts of auxiliary substances such as wetting agents or emulsifying agents, preservatives or buffers, which desirably can enhance the shelf life or effectiveness of the antibody molecule, related composition, or combi- nation. Suitability for carriers and other components of pharmaceutical compositions is determined based on the lack of significant negative impact on the desired biological properties of the antibody molecule, related composition, or combination (e.g., less than a substantial impact (10% or less relative inhibition, 5% or less relative inhibition, etc.) on G1/G2 and secondary target binding) In general, antibody molecules and related compositions can be administered in combination with any suitable pharmaceutically acceptable excipient or combination thereof (i.e., any suitable excipient component). A pharmaceutically acceptable excipient refers to any inactive agent that is combined with an active agent to form a pharmaceutically acceptable and active composition. Excipients include inert pharmaceutically acceptable carriers and diluents. Excipients also include compositions that modulate (and typically improve) the physiochemical properties of a pharmaceutical composition. Examples of such excipients include stabilizers, preservatives, solubilizers, solvents, and solutes. Excipients also include flavorants, coloring agents, etc. Antibody molecules and related compounds and combinations described herein can be formulated in any manner suitable for administration to a sub- ject, such as a human patient.
In one such aspect, an antibody molecule can be combined with one or more carriers appropriate a desired route of administration, antibody molecules may be, for example, admixed with lactose, sucrose, powders (e.g., starch powder), cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and optionally further tabletted or encapsulated for conventional administration. Alternatively, an antibody or other antibody molecules may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buff- ers. Other carriers, adjuvants, and modes of administration are well known in the pharmaceutical arts. A carrier may include a time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.
Pharmaceutically acceptable carriers generally include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption de- laying agents, and the like that are physiologically compatible with an antibody molecule or related composition or combination provided by the invention. Further examples of pharmaceutically acceptable carriers include sterile water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, and the like, as well as combinations of any thereof.
In many cases, it can be desirable to include isotonic agents, for example, sugars, polyalcohols (such as mannitol), sorbitol, or sodium chloride in a pharmaceutical composition. Pharmaceutically acceptable substances such as wetting agents, emulsifying agents, preservatives, and buffers, which desirably can enhance the shelf life or effectiveness of the antibody molecule and/or related composition active component.
Suitability for carriers and other components of pharmaceutical compositions is typi- cally determined based on the lack of significant negative impact on the desired biological properties of the antibody molecule, related composition, or combination (e.g., less than a substantial impact - such as about 10% or less relative inhibition, about 5% or less relative inhibition, etc. on G1/G2 binding by the antibody molecule component of the composition). See also, e.g., Powell et al. "Compendium of excipients for parenteral formulations" PDA J Pharm Sci Technol. 52:238-31 1 (1998) and the citations therein for additional information related to typically suitable excipients well known to pharmaceutical chemists.
A composition for pharmaceutical use also or alternatively can include various diluents, fillers, salts, buffers, detergents (e.g., a nonionic detergent, such as Tween-80), stabi- lizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and/or other materials suitable for inclusion in a composition for pharmaceutical use. Examples of suitable components also are described in, e.g., Berge et al., J. Pharm. Sci., 6661 ), 1 - 19 (1977); Wang and Hanson, J. Parenteral. Sci. Tech: 42, S4-S6 (1988);US Patents 6,165,779 and 6,225, 289; and other documents cited herein. Such a pharmaceutical com- position also can include preservatives, antioxidants, or other additives known to those of skill in the art. Additional pharmaceutically acceptable carriers are known in the art and described in, e.g., Urquhart et al., Lancet, 16, 367 (1980), Lieberman et al., Pharmaceutical Dosage Forms-Disperse Systems (2nd ed., vol. 3,1998); Ansel et al., PHARMACEUTICAL DOSAGE FORMS & DRUG DELIVERY SYSTEMS (7th ed. 2000); Martindale, THE EXTRA PHARMA- COPEiA (31 st edition), REMINGTON'S PHARMACEUTICAL SCIENCES (16th-20th editions); The PHARMACOLOGICAL BASIS OF THERAPEUTICS, Goodman and Gilman, Eds. (9th ed.-1996); Wilson and Gisvolds1 TEXTBOOK OF ORGANIC MEDICINAL AND PHARMACEUTICAL CHEMISTRY, Delgado and Remers, Eds. (10th ed. -1998), and U.S. Patents 5,708,025 and 5,994,106. Principles of formulating pharmaceutically acceptable compositions also are de- scribed in, e.g., Platt, Clin. Lab Med., 7:289-99 (1987), Aulton, PHARMACEUTICS: THE SCIENCE OF DOSAGE FORM DESIGN, Churchill Livingstone (New York) (1988), EXTEMPORANEOUS ORAL LIQUID DOSAGE PREPARATIONS, CSHP (1998), and "Drug Dosage," J. Kans. Med. Soc, 70 (I), 30-32 (1969). Further additional pharmaceutically acceptable carriers particularly suitable for administration of antibody molecule compositions and related composi- tions (e.g., compositions comprising antibody molecule-encoding nucleic acids or antibody molecule-encoding nucleic acid comprising vectors) are described in, e.g., WO 98/32859. Antibody molecule compositions also include compositions comprising any suitable combination of an antibody molecule peptide and related salt. Any suitable salt, such as an alkaline earth metal salt in any suitable form (e.g., a buffer salt), can be used in the stabiliza- tion of antibody molecules (preferably the amount of salt is such that oxidation and/or precipitation of the antibody molecule is avoided). Suitable salts typically include sodium chloride, sodium succinate, sodium sulfate, potassium chloride, magnesium chloride, magnesium sulfate, and calcium chloride. In one aspect, an aluminum salt is used to stabilize an antibody molecule in a composition of the invention, which aluminum salt also may serve as an adju- vant when such a composition is administered to a patient. Compositions comprising a base and antibody molecules also are provided. In other aspects, the invention provides an antibody molecule composition that essentially lacks a tonicifying amount of any salt.
Antibody molecule compositions, related compositions, and combinations according to the invention may be in a variety of suitable forms. Such forms include, for example, Nq- uid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, emulsions, microemulsions, tablets, pills, powders, liposomes, dendrimers and other nanoparticles (see, e.g., Baek et al., Methods Enzymol. 2003;362:240-9; Nigavekar et al., Pharm Res. 2004 Mar;21 (3):476-83), microparticles, and suppositories. In one aspect, the invention provides an effective amount of an anti-α3 G1 G2 antibody contained in liposomes formulated for delivery to cancer-associated cells.
Formulations of antibody molecule compositions also can include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles, DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions, carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present invention, provided that the binding of the antibody molecule to α3 is not significantly inhibited and/or the biological activity of related molecule(s) significantly inhibited by the formulation and the formulation is physiologically compatible and tolerable with the planned route of administration. The op- timal form for any antibody molecule-associated composition depends on the intended mode of administration, the nature of the composition or combination, and therapeutic application or other intended use.
Typically, compositions in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies, are used for delivery of antibody molecules of the invention. A typical mode for delivery of antibody molecule compositions is by parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, and/or intramuscular administration). In one aspect, an anti-α3 G1 G2 antibody is administered to a human patient by intravenous infusion or injection. In another aspect, an anti-α3 G1 G2 antibody is administered by intramuscular or subcutaneous injec- tion. As already indicated, intratumor administration also may be useful in certain regimens. Antibody molecules, such as anti-α3 G1 G2 antibodies, antibody fragments, and derivatives thereof, also may be formulated in, for example, solid formulations (including, e.g., granules, powders, projectile particles, or suppositories), semisolid forms (gels, creams, etc.), or in liquid forms (e.g., solutions, suspensions, or emulsions). Antibodies and other antibody molecules also may be applied in a variety of solutions (e.g., an aqueous solution). Suitable solutions for use in accordance with the invention typically are sterile, dissolve sufficient amounts of the antibody and other components (e.g., an immunomodulatory cytokine such as GM-CSF, IL-2, and/or KGF), stable under conditions for manufacture and storage, and not harmful to the subject for the proposed application.
An antibody molecule may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc. A composition also can be formulated as a solution, microemulsion, dispersion, powder, macroemulsion, liposome, or other ordered structure suitable to high drug concentration. Desirable fluidity properties of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. These and other components of a pharmaceutically acceptable composition of the invention can impart advantageous properties such as improved transfer, delivery, tolerance, and the like.
In one exemplary aspect, an antibody molecule is prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, poly- glycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., SUSTAINED AND CONTROLLED RELEASE DRUG DELIVERY SYSTEMS, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Other teachings relevant to such systems are known in the art. For example, Heller, Biodegradable Polymers in Controlled Drug Delivery, in: CRC Critical Reviews in Therapeutic Drua Carrier Systems, Vol. 1 , CRC Press, Boca Raton, FIa., 1987, pp 39-90, describes encapsulation for controlled drug delivery, and Di Colo (1992) Biomaterials 13:850-856 describes controlled drug release from hydrophobic polymers.
In another aspect, compositions of the invention orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
Antibody molecules, related compounds (e.g., antibody molecule-encoding nucleic acids), and related compositions can generally be delivered in any suitable dosage. Deter- mination of precise dosage for optimal effect will vary with a number of factors (condition to be treated, age of patient, health of patient, type(s) of antibody molecule or surrogate used, presence of additional active agents and/or application of related therapies, etc.), such that it is often more useful to describe dosage in terms of an amount sufficient or optimal for inducing, promoting, and/or enhancing a particular effect. In this respect, compositions of the in- vention can include "therapeutically effective amount," a "prophylactically effective amount", or "physiologically effective amount" of an antibody molecule or related composition (or "first" and "second" amounts in the case of a combination composition comprising an antibody molecule and a second element; first, second, and third amounts in the case of three included agents; etc.). A "therapeutically effective amount" refers to an amount effective, when delivered in appropriate dosages and for appropriate periods of time, to achieve a desired therapeutic result in a host (e.g., the inducement, promotion, and/or enhancement of a physiological response associated with reducing one or more aspects of cancer progression, increasing the likelihood of survival over a period of time (e.g., 18-60 months after initial cancer treatment), reducing the spread of cancer cell-associated growths, and/or reducing the likelihood of recurrence of tumor growth). A therapeutically effective amount of an antibody molecule may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody molecule to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the anti- body or antibody portion are outweighed by the therapeutically beneficial effects.
A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result (e.g., a reduction in the likelihood of developing a disorder, a reduction in the intensity or spread of a disorder, an increase in the likelihood of survival during an imminent disorder, a delay in the onset of a dis- ease condition, a decrease in the spread of an imminent condition as compared to in similar patients not receiving the prophylactic regimen, etc.). Typically, because a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
Where the phrase "effective amount" is used without a modifier such as "therapeuti- cally" or "prophylactically", the phrase is intended to mean an amount that is at least as great as the minimum prophylactically effective or therapeutically effective amount and that is appropriate for the indicated use. In one exemplary aspect, the invention relates to a method of treating cancer in a patient comprising delivering to the patient an effective amount of one or more antibody molecules, an antibody molecule combination composition, a related composi- tion, etc. Such an amount typically ranges from the minimum amount required to induce an effective prophylactic effect in a patient that is not suffering from a cancer condition (but may be at risk of developing such a condition) to an amount sufficient for one or more therapeutic regimens (e.g., significantly reducing tumor burden). In other words, the phrase "effective amount" encompasses both "prophylactically effective" and "therapeutically effective" amounts unless otherwise stated or clearly contradicted by context.
Antibody molecules can be combined with a large number of anti-cancer therapeutic and/or prophylactic agents and therapies. Non-limiting examples of such "second" or "secondary" agents that can be included in a composition with an antibody molecule ("combination compositions") include fluoropyrimidiner carbamates, such as capecitabine; non- polyglutamatable thymidylate synthase inhibitors; nucleoside analogs, such as tocladesine; antifolates such as pemetrexed disodium; taxanes and taxane analogs; topoisomerase inhibitors; polyamine analogs; mTOR inhibitors (e.g., rapamcyin ester); alkylating agents (e.g., oxaliplatin); lectin inhibitors; vitamin D analogs (such as seocalcitol); carbohydrate processing inhibitors; antimetabolism folate antagonists; thumidylate synthase inhibitors; other anti- metabolites (e.g., raltitrexed); ribonuclease reductase inhibitors; dioxolate nucleoside analogs; thimylate syntase inhibitors; gonadotropin-releasing hormone (GRNH) peptides; human chorionic gonadotropin; chemically modified tetracyclines (e.g., CMT-3; COL-3); cytosine deaminase; thymopentin; DTIC; carmustine; carboplatin; vinblastine; temozolomide; vinde- sine; thymosin-α; histone deacetylase inhibitors (e.g., phenylbutyrate); DNA repair agents (e.g., DNA repair enzymes and related compositions such as Dimericine™ (T4 endonuclease V-containing liposome)); gastrin peptides (and related compositions such as Gastrim- mune™); GMK and related compounds/compositions (see, e.g., Knutson, Curr Opin Investig Drugs. 2002 Jan;3(1 ):159-64 and Chapman et al., Clin Cancer Res. 2000 Dec;6(12):4658- 62); beta-catenin blockers/inhibitors and/or agents that lower the amount of beta-catenin production in preneoplastic or neoplastic cell nuclei (see, e.g., US Patent 6,677,1 16), agents that upregulate E-cadherin expression (or E-cadherin); agents that reduce slug (beta- catenin-associated) gene expression; agents that block, inhibit, or antagonize PAI-1 or that otherwise modulate urokinase plasminogen activator (uPA) interaction with the uPA receptor; survivins; DNA demethylating agents; "cross-linking" agents such as platinum-related anti- cancer agents (cisplatin, carboplatin, etc.); agents that block antiapoptotic signaling, such as agents that inhibit MAPK and Ras signaling pathways or components thereof (e.g., agents that interfere with the production and/or function of cyclin D); growth suppressive agents, such as an antimetabolite such as Cepecitabine/Xeloda, cytarabine/Ara-C, Cladrib- ine/Leu statin, Fludaraine/Fludara, fluorouracil/5-FU, gemcitabine/Gemzar, mercaptopurine/6- MP, methotrexate/MTX, thioguanine/6-TG, Allopurinol/Zyloprim, etc.; an acylating agent such as Busulfan, Cyclophosphamide, mechlorethamaine, Melphalan, thiotepa, semustine, car- boplatin, cisplatin, procarbazine, dacarbazine, Althretamine, Lomustine, Carmustine, Chlorambucil, etc.; a topoisomerase inhibitor such as Camptothecins as Topotecan, Iri- notecan; such as Podophyllotoxins as Etoposide/VP16, Teniposide/VP26, etc.; an inhibitor of microtuble and/or spindle formation, such as Vincristine, Vinblastine, Vinorelbine, or Taxane such as Paxlitaxel, Docetaxel, combrestatin, Epothilone B, etc; RRR-alpha-tocopheryl succinate; anthracyclins as Daunorubicin/Cerubidine and Doxorubicin; idarubicin; mitomycins; pli- camycin; retinoic acid analogues such as all trans retinoic acid, 13-cis retinoic acid, etc.; inhibitors of receptor tyrosine kinases; inhibitors of ErbB-1/EGFR such as iressa, Erbitux, etc.; inhibitors of ErbB-2/Her2 such as Herceptin, etc.; inhibitors of c-kit such as Gleevec; inhibitors of VEGF receptors such as ZD6474, SU6668, etc.; Inhibitors of ErbB3, ErbB4, IGF-IR, insulin receptor, PDGFRa, PDGFRbeta, Flk2, Flt4, FGFR1 , FGFR2, FGFR3, FGFR4, TRKA, TRKC, c-met, Ron, Sea, Tie, Tie2, Eph, Ret, Ros, AIk, LTK, PTK7, etc.; cancer related enzyme inhibitors such as metalloproteinase inhibitors such as marimastat, Neovastat, etc.; cathepsin B; modulators of cathepsin D dehydrogenase activity; glutathione-S-transferases and related compounds such as glutacylcysteine synthetase and lactate dehydrogenase; proteasome inhibitors (e.g., Bortezomib); tyrosine kinase inhibitors; farnesyl transferase inhibitors; HSP90 inhibitors (e.g., 17-allyl amino geld-anamycin) and other heat shock protein- inhibitors; mycophenolate mofetil; mycophenolic acid; asparaginase; calcineurin-inhibitors; TOR-inhibitors; multikine molecules; enkephalins (see, e.g., US Patent 6,737,397); SU1 1248 (Pfizer); BAY 43-9006 (Bayer and Onyx); inhibitors of "lymphocyte homing" mechanisms such as FTY720; Tarceva; Iressa; Glivec; thalidomide; and adhesion molecule inhibitors (e.g., anti-LFA, etc.). Additional anti-neoplastic agents that can be used in the combination composition and combination administration methods of the invention include those de- scribed in, e.g., US Patents 6,660,309, 6,664,377, 6,677,328, 6,680,342, 6,683,059, and 6,680,306, as well as International Patent Application WO 2003070921 .
In one aspect, the invention provides combination compositions and combination delivery/administration protocols that include one or more biological response modifiers (BRMs) in addition to one or more antibody molecules. BRMs generally are products pro- duced by cells that stimulate or restore the ability of the immune system to act against disease agents (e.g., cancer cells), such as cytokines or antibodies.
In a particular aspect, the invention provides a combination composition that includes at least one antibody molecule and at least one secondary anti-cancer monoclonal antibody. A number of suitable anti-cancer mAbs are known in the art and similar suitable antibodies can be developed against cancer-associated targets. Particular examples of suitable second anti-cancer mAbs include anti-CD20 mAbs (such as Rituximab and HuMax- CD20), anti-Her2 mAbs (e.g., Trastuzumab), anti-CD52 mAbs (e.g., Alemtuzumab and Ca- path® 1 H), anti-EGFR mAbs (e.g., Cetuximab, HuMax-EGFr, and ABX-EGF), Zamyl, Pertu- zumab, anti-A33 antibodies (see US Patent 6,652,853), anti-oncofetal protein mAbs (see US Patent 5,688,505), anti-PSMA mAbs (see, e.g., US Patent 6,649,163 and Milowsky et al., J Clin Oncol. 2004 Ju1 1 ;22(13):2522-31 . Epub 2004 Jun 01 ), anti-TAG-72 antibodies (see US Patent 6,207,815), anti-aminophospholipid antibodies (see US Patent 6,406,693), anti- neurotrophin antibodies (US Patent 6,548,062), anti-C3b(i) antibodies (see US Patent 6,572,856), anti-cytokeratin (CK) mAbs, anti-MN antibodies (see, e.g., US Patent 6,051 ,226), anti-mts1 mAbs (see, e.g., US Patent 6,638,504), anti-PSA antibodies (see, e.g., Donn et al., Andrologia. 1990;22 Suppl 1 :44-55; Sinha et al., Anat Rec. 1996 Aug;245(4):652-61 ; and Katzenwadel et al., Anticancer Res. 2000 May-Jun;20(3A):1551 -5); antibodies against CA125; antibodies against integrins like integrin betai ; antibodies/inhibitors of VCAM; anti- alpha-v/beta-3 integrin mAbs; anti-kininostatin mAbs; anti-aspartyl (asparaginyl) beta- hydroxylase (HAAH) intrabodies (see, e.g., US Patent 6,783,758); anti-CD3 mAbs (see, e.g., US Patents 6,706,265 and 6,750,325) and anti-CD3 bispecific antibodies (e.g., anti-CD3/Ep- CAM, anti-CD3/her2, and anti-CD3/EGP-2 antibodies - see, e.g., Kroesen et al., Cancer Immunol Immunother. 1997 Nov-Dec;45(3-4):203-6); and anti-VEGF mAbs (e.g., bevacizu- mab). Other possibly suitable second mAb molecules include alemtuzumab, edrecolomab, tositumomab, ibritumomab tiuxetan, and gemtuzumab ozogamicin. In one aspect, the invention provides combination compositions and combination therapies that comprise one or more antibodies, typically monoclonal antibodies, targeted against angiogenic factors and/or their receptors, such as VEGF, bFGF, and angiopoietin-1 ; and monoclonal antibodies against other relevant targets (see also, generally, Reisfeld et al., lnt Arch Allergy Immunol. 2004 Mar;133(3):295-304; Mousa et al., Curr Pharm Des. 2004;10(1 ):1 -9; Shibuya, Nippon Yakurigaku Zasshi. 2003 Dec;122(6):498-503; Zhang et al., MoI Biotechnol. 2003 Oct;25(2):185-200; Kiselev et al., Biochemistry (Mosc). 2003 May;68(5):497-513; Shepherd, Lung Cancer. 2003 Aug;41 Suppl 1 :S63-72; O'Reilly, Methods MoI Biol. 2003;223:599-634; Zhu et al., Curr Cancer Drug Targets. 2002 Jun;2(2):135-56; and WO 2004/035537). In another particular facet, the invention provides combination compositions and combination therapies that involve effective amount(s) of one or more antibody molecules and one or more chemotherapeutic agents selected from 5-Fluorouracil, actinomycin D (Dactinomycin), amsacrine, arsenic trioxide, asparaginase, azadcytadine (5-azacytidine, 5AZ), busulfan (myleran), capecitabine, carboplatin (paraplatin), carmustine (BiCNU), chlorambucil (Leukeran), cisplatin (Platinol), cyclophosphamide (Cyroxan), cytarabine (Ara- C), Dacarbazine, Dactinomycin, Daunorubicin (Cerubidine), Docetaxel (Taxotere), Doxorubicin (Adriamycin Doxil), Epirubicin (Ellence), Etoposide (VP-16, Vespid), Fludarabine, Fluorouracil, Gemcitabine, Gleevac (Imatinib mesylate), Hydroxyurea, Idarubicin, lfosfamide (Ifex), Irinotecan, Liposomal Doxurubicin, Lomustine, Mechlorechamine (Mustargen), MeI- phalan, Mercaptopurine, Methotrexate, Methyl CCNU, Mitomycin (Mutamycin), Mitoxantrone, Nitrogen Mustard, Nitrosoureas, Oxaliplatin, Paclitaxel (Taxol), Pentostatin, Plicamycin, Procarbazine, Streptozocin, Telcyta (alone or in combination with Doxil), Teniposide (Vumon), Thiotepa, Tretinoin, Vinblastine (Velban), Vincristine (Oncovin), and Vinorelbine (Navelbine). To better illuminate the invention, non-limiting groups of chemotherapeutic agents useful in antibody molecule combination compositions and therapies are described here. In one aspect, the invention provides combination compositions and combination therapies characterized in comprising one or more alkylating agents. Alkylating agents are so named because of their ability to add alkyl groups to many electronegative groups under conditions present in cells. These agents stop tumor growth by cross-linking guanine bases in DNA double-helix strands - directly attacking DNA. This makes the strands unable to uncoil and separate. As this is necessary in DNA replication, cells affected by such agents can no longer divide. Examples of alkylating agents include Busulfan (Myleran), Busulfan Injection (Busulfex Injection), Carboplatin (Paraplatin), Carmustine Injection (BiCNU Injection), Chlorambucil (Leukeran), Cyclophosphamide Injection (Cytoxan Injection, Neosar), Dacarbazine (DTIC, DTIC-Dome), lfosfamide (Ifex), Lomustine (CCNU, CeeNU), Mechlorethamine (Mustargen, Nitrogen mustard), Melphalan (Alkeran, L-PAM), Melphalan Injection (Alkeran Injection), Streptozocin (Zanosar), and Thiotepa (Thioplex).
In another facet, the invention provides combination compositions and therapies that comprise an antibody molecule and an antimetabolite. Antimetabolites prevent cancer cells from processing nutrients and other substances that are necessary for normal activity in the cancer cells. More particularly, antimetabolites masquerade as purine or pyrimidine, which become the building blocks of DNA. They prevent these substances becoming incorporated in to DNA during the "S" phase of the cell cycle, thereby preventing cell division. There are several different cellular targets for antimetabolites. Some common classes of antimetabolites Folate Antagonists, Purine Antagonists, and Pyrimidine Antagonists.
Folate antagonists, also known as antifolates, inhibit dihydrofolate reductase (DHFR), an enzyme involved in the formation of nucleotides. When this enzyme is blocked, nucleotides are not formed, disrupting DNA replication and cell division. Methotrexate is the primary folate antagonist used as a chemotherapeutic agent.
The purine antagonists function by inhibiting DNA synthesis in two different ways. They can inhibit the production of the purine containing nucleotides, adenine and guanine. If a cell does not have sufficient amounts of purines, DNA synthesis is halted and the cell can- not divide. They also may be incorporated into the DNA molecule during DNA synthesis. The presence of the inhibitor is thought to interfere with further cell division. Examples of purine antagonists include 6-Mercaptopurine, Dacarbazine, and Fludarabine.
Pyrimidine antagonists also can be combined or co-delivered with one or more antibody molecules. Pyrimidine antagonists act to block the synthesis of pyrimidine containing nucleotides. These drugs typically have structures similar to the natural compound that they replace. By acting as 'decoys', these drugs can prevent the production of the finished nucleotides. They may exert their effects at different steps in that pathway and may directly inhibit crucial enzymes. Pyrimidine antagonists may also be incorporated into a growing DNA chain and lead to termination of the process. Examples of pyrimidine antagonists in- elude 5-fluorouracil, Arabinosylcytosine, Capecitabine, and Gemcitabine.
In an exemplary aspect, the invention provides combination compositions and therapies characterized by including one or more metabolites selected from Floxuridine (FUDR, Fluorodeoxyuridine), Fludarabine Phosphate (Fludara), Gemcitabine Hydrochloride (Gemzar), Hydroxyurea (Droxia, Hydrea), Mercaptopurine (6-MP, Purinethol), Methotrexate (Rheumatrex, Trexall), Methotrexate Injection (Amethopterin, MTX Injection), Thioguanine (6- TG, TG), and combinations of any thereof.
The invention also relates to combination compositions and therapies including one or more antineoplastic hormones. Antineoplastic hormones interfere at the cellular level with receptors for growth stimulating proteins. By blocking the receptor, the production or release of growth factors is reduced. Examples of such agents include Diethylstilbestrol Injection (Stilphostrol Injection), Megestrol (Megace), and Mitotane (Lysodren).
In a further facet, the invention provides combination compositions and combination delivery methods characterized in including one or more mitotic inhibitors. Mitotic inhibitors generally prevent cell division by interfering with the protein called tubulin. Tubulin is the ba- sic building block of the fibers that are responsible for ensuring that each cell continues to multiply. Examples of such agents include Docetaxel (Taxotere), Etoposide Injection (To- posar, VePesid Injection), Etoposide Oral (VP-16, VePesid Oral), Paclitaxel (Onxol, Taxol), Vinblastine (Velban), and Vincristine (Oncovin, Vincasar).
Another somewhat related class of classic chemotherapeutic agents is plant alka- loids. These alkaloids are derived from plants and block cell division by preventing microtubules being synthesized. These are vital for cell division and without them, it can not occur. The main examples are vinca alkaloids such as vincristine.
Another also somewhat related class of classic chemotherapeutic agents is "genotoxic drugs." Genotoxic drugs are chemotherapy agents that affect nucleic acids and alter their function. These drugs may directly bind to DNA or they may indirectly lead to DNA damage by affecting enzymes involved in DNA replication and possibly apoptosis. Such genotoxic chemotherapy treatments include alkylating agents, intercalating agents (drugs that "wedge" into the spaces between the nucleotides in the DNA double helix, thereby interfering with transcription and replication, and often inducing mutations), and enzyme inhibitors (e.g., agents that inhibit key enzymes, such as topoisomerases, involved in DNA replication inducing DNA damage). Thus, the genotoxic drug class of chemotherapeutic agents overlaps with other classes described elsewhere herein. A goal of treatment with any of these agents includes (i.e., a common mechanism of action associated with such agents is) the induction of DNA damage in the cancer cells. DNA damage, if severe enough, will induce cells to undergo apoptosis. Genotoxic chemotherapy drugs typically affect both normal and cancerous cells. The selectivity of the drug action is based on the sensitivity of rapidly dividing cells, such as cancer cells, to treatments that damage DNA. Examples of agents that can be classified as genotoxic agents include Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Daunorubicin, Doxorubicin, Epirubicin, Etoposide, Idarubicin, Ifosfamide, Irinotecan, Lomustine, Mechlorethamine, Melphalan, Mitomycin C, Mitoxantrone, Oxaliplatin, Temozolamide, and Topotecan.
Another feature of the invention is embodied in combination methods and compositions that comprise one or more antibody molecules (or delivery thereof) and one or more nucleic acids that act as anti-cancer agents (or delivery thereof). In a particular aspect, the invention provides combination compositions and methods, wherein an antibody molecule is combined with or delivered in association with a nucleic acid comprising a sequence encoding a tumor suppressor. In one exemplary facet, one or more antibody molecules are delivered in association with the delivery of a p53 tumor suppressor gene (see, e.g., Roth et al., Oncology (Huntingt). 1999 Oct;13(10 Suppl 5):148-54) and Nielsen et al., Cancer Gene Ther. 1998 Jan-Feb;5(1 ):52-63). Additional tumor suppres- sor targets include, for example, BRCA1 , RB1 , BRCA2, DPC4 (Smad4), p21 , E2F-1 , FUS1 compounds (e.g., INGN 401 ), MSH2, MLH1 , and DCC. Such a nucleic acid can be delivered in the form of a suitable vector, host cell, etc. For example, one or more antibody molecules can be combined with or delivered in association with a replication-deficient adenovirus en- coding human recombinant wild-type p53/SCH58500.
A further feature of the invention is combination methods and combination compositions that include one or more antibody molecules and one or more nucleic acids that are able to reduce one or more aspects of expression of particular cancer-associated genes. Such agents include antisense nucleic acids and inhibitory RNA (iRNA) molecules. In one exemplary aspect, the invention provides combination compositions and methods that involve one or more antisense nucleic acids targeted to oncogenes, mutated, or deregulated genes. In another exemplary aspect, the invention provides combination compositions and methods that involve at least one siRNA molecule targeted to mutated or deregulated genes. Another feature of the invention is combination compositions and meth- ods that include one or more antisense oligonucleotides and/or siRNAs that reduce the expression of oncogenes or other cancer progression-related genes (e.g., Ln5-targeted or in- tegrin-targeted antisense molecules - which are described in, e.g., US Patent Application 2003224993 and O'Toole et al., Exp Cell Res. 1997 Jun 15;233(2):330-9, and/or antisense molecules against Ln5-modulators, such as MT 1 -MMP antisense oligonucleotides (see, e.g., Giles et al., J Cell Sci. 2001 Aug;1 14(Pt 16):2967-76)), other inhibitors of Ln5 production or activity (e.g., p300 - see, e.g., Miller et al., J Biol Chem. 2000 Mar 17;275(1 1 ):8176-82), mutated genes, and/or deregulated genes.
For example, an antibody molecule, such as an anti-α3 G1 G2 antibody or fragment thereof, can be combined with or administered in association with an anti-cancer antisense nucleic acid (e.g., Genasense™ (augmerosen/G3139)), LY900003 (ISIS 3521 ), ISIS 2503, OGX-01 1 (ISIS 1 12989), LE-AON/LEraf-AON (liposome encapsulated c-raf antisense oli- gonucleotide/ISIS-5132), MG98, and other antisense nucleic acids that target PKCα, clus- terin, IGFBPs, protein kinase A, cyclin D1 , or Bcl-2 - see, e.g., Benimetskaya et al., Clin Prostate Cancer. 2002 Jun;1 (1 ):20-30; Tortora et al., Ann N Y Acad Sci. 2003 Dec;1002:236- 43; Gleave et al., Ann N Y Acad Sci. 2003 Dec;1002:95-104.; Lahn et al., Ann N Y Acad Sci. 2003 Dec;1002:263-70; Kim et al., lnt J Oncol. 2004 Jan;24(1 ):5-17; Stahel et al., Lung Cancer. 2003 Aug;41 Suppl 1 :S81 -8; Stephens et al., Curr Opin MoI Ther. 2003 Apr;5(2):1 18-22; Cho-Chung, Arch Pharm Res. 2003 Mar;26(3):183-91 ; and Chen, Methods MoI Med. 2003;75:621 -36)). In another exemplary aspect, an antibody molecule is delivered in asso- ciation with or combined in a composition with an anti-cancer inhibitory RNA molecule (see, e.g., Lin et al., Curr Cancer Drug Targets. 2001 Nov;1 (3):241 -7, Erratum in: Curr Cancer Drug Targets. 2003 Jun;3(3):237; Lima et al., Cancer Gene Ther. 2004 May;1 1 (5):309-16; Grzmil et al., lnt J Oncol. 2004 Jan;24(1 ):97-105; CoIMs et al., lnt J Radiat Oncol Biol Phys. 2003 Oct 1 ;57(2 Suppl):S144; Yang et al., Oncogene. 2003 Aug 28;22(36):5694-701 ; and Zhang et al., Biochem Biophys Res Commun. 2003 Apr 18;303(4):1 169-78 for discussion relating to such iRNA molecules, related principles, and related methods).
In another facet, the invention provides combination compositions and combination administration methods where an α3 G1 G2-antibody molecule is combined with an anticancer nucleozyme, such as a ribozyme, examples of which include angiozyme (Ribozyme Pharmaceuticals) (see e.g., Pennati et al., Oncogene. 2004 Jan 15;23(2):386-94; Tong et al., Clin Lung Cancer. 2001 Feb;2(3):220-6; Kijima et al., lnt J Oncol. 2004 Mar;24(3):559-64; Tong et al., Chin Med J (Engl). 2003 Oct;1 16(10):1515-8; and Orlandi et al., Prostate. 2003 Feb 1 ;54(2):133-43) and herzyme (in a related sense see US Patent 6,617,438). Additional anti-cancer ribozymes are described in, e.g., US Patent Applications 20030195164, 20030050236, and 20030105043 and US Patents 6,482,803 and 6,489,163. See also Polis- eno et al., Current Pharmaceutical Biotechnology August 2004, vol. 5, no. 4, pp. 361 -368(8) for a review of RNA-based drugs.
In yet another aspect, an antibody molecule is combined or co-delivered with an immunostimulatory nucleic acid (in another aspect, a nucleic acid comprising a sequence encoding an antibody molecule and at least one immunostimulatory sequence is provided). Numerous examples of suitable immunostimulatory nucleic acids have been described in the art (see, e.g., Krieg, Trends in Microbiol 7: 64-65 (1999); Wooldridge et al., Curr Opin Oncol. 2003 Nov;15(6):440-5; Jahrsdorfer et al., Semin Oncol. 2003 Aug;30(4):476-82; Jahrsdorfer et al., Curr Opin Investig Drugs. 2003 Jun;4(6):686-90; Carpentier et al., Front Biosci. 2003 Jan 1 ; 8:e1 15-27; US Patent 6,406,705; US Patent 6,218,371 ; US Patent Application 20040181045; and US Patent Application 20040087538).
In another aspect, the invention provides methods for treating α3-associated conditions that comprising delivering one or more antibody molecules and/or combining one or more antibody molecules (or related compositions) with at least one Ln5-encoding nucleic acid (in a form associated with normal cell basement membrane attachment/association), at least one nucleic acid that upregulates endogenous Ln5 production (e.g., by so-called gene activation), and/or one or more cells expressing Ln5 at levels at least as great as in normal basement membrane-associated epithelial cells. Other functional gene replacement methods also can be used in the context of the methods and reflected in compositions of this in- vention (e.g., providing a nucleic acid encoding a non-cancer-associated version of a tumor suppressor such as p53). Another use of gene therapy is the introduction of enzymes into these cells that make cancer cells susceptible to particular chemotherapy agents (e.g., introducing thymidine kinase into cancer cells so as to make them susceptible to aciclovir).
In a further aspect, the invention provides combination compositions and administra- tion methods wherein an antibody molecule is combined with or co-administered with a basal lamina-targeted and/or basal lamina-associated factor modulating anti-cancer molecules (e.g., a molecule that inhibits breakdown of the basal lamina in cancer progression), such as ginsenoside-Rb2, anti-MMP-1 antibodies, anti-integrin antibodies, anti-MMP2 antibodies and inhibitors, anti-MT1 -MMP antibodies and inhibitors, anti-EGF-R antibodies, anti-BMP-1 inhibi- tors and antibodies, and inhibitors of urokinase-type plasminogen activator (uPA) and/or plasminogen activation to plasmin (aprotinin, amiloride, EACA, tranexamic acid, anti-uPAn antibody). In another aspect, the invention provides such compositions and methods wherein the composition or method comprises an inhibitor of Thymosin beta 4.
In an additional facet, the invention provides combination compositions and combi- nation administration methods ("combination therapies") wherein an antibody molecule is combined or codelivered with a virus or related molecule (e.g., a virus like particle, a viral nucleic acid, etc.) that acts as an active agent against cancer. In one aspect, the invention provides combination compositions and methods that comprise one or more antibody molecules (or related compositions) and at least one oncolytic virus. Examples of such viruses include oncolytic adenoviruses and anti-cancer herpes viruses, which may or may not be modified viruses (see, e.g., Teshigahara et al., J Surg Oncol. 2004 Jan;85(1 ):42-7; Stiles et al., Surgery. 2003 Aug;134(2):357-64; Zwiebel et al., Semin Oncol. 2001 Aug;28(4):336-43; Varghese et al., Cancer Gene Ther. 2002 Dec;9(12):967-78; and Wildner et al., Cancer Res. 1999 Jan 15;59(2):410-3). Various viruses, viral proteins, and the like can be used in combination compositions and combination administration methods. Replication-deficient viruses, that generally are capable of one or only a few rounds of replication in vivo, and that are targeted to tumor cells, can, for example, be useful components of such compositions and methods. Such viral agents can comprise or be associated with nucleic acids encoding immunostimulants, such as GM-CSF and/or IL-2. Both naturally oncolytic and such recombinant oncolytic viruses (e.g., HSV-1 viruses; reoviruses; replication-deficient and replication-sensitive adenovirus; etc.) can be useful components of such methods and compositions (see, e.g., Varghese et al., Cancer Gene Ther. 2002 Dec;9(12):967-78; Zwiebel et al., Semin Oncol. 2001 Aug;28(4):336-43; Sunarmura et al., Pancreas. 2004 Apr;28(3):326-9; Shah et al., J Neu- rooncol. 2003 Dec;65(3):203-26; and Yamanaka, lnt J Oncol. 2004 Apr;24(4):919-23). Additional features of the invention include combination administration methods and combination compositions wherein an antibody molecule is combined or delivered with an anti-cancer immunogen, such as a cancer antigen/tumor-associated antigen (e.g., an epithelial cell adhesion molecule (Ep-CAM/TACSTDI ), mucin 1 (MUC1 ), carcinoembryonic antigen (CEA), tumor-associated glycoprotein 72 (TAG-72), gp100, Melan-A, MART-1 , KDR, RCAS1 , MDA7, CEA, cyclin-dependent kinase 4, β-catenin, capsase-B, tyrosinase, cancer- associated viral vaccines (e.g., human papillomavirus vaccines), tumor-derived heat shock proteins, and the like, additional examples of which are described elsewhere herein) (see also, e.g., Acres et al., Curr Opin MoI Ther 2004 Feb, 6:40-7; Taylor-Papadimitriou et al., Biochim Biophys Acta. 1999 Oct 8;1455(2-3):301 -13; Emens et al., Cancer Biol Ther. 2003 Jul-Aug;2(4 Suppl 1 ):S161 -8; and Ohshima et al., lnt J Cancer. 2001 Ju1 1 ;93(1 ):91 -6). A number of other suitable cancer antigens/tumor-associated antigens described herein (e.g., gp75) and similar molecules known in the art also or alternatively can be used in such combination administration methods or incorporated in such combination compositions. Anti-cancer immunogenic peptides also include anti-idiotypic "vaccines" such as
BEC2 anti-idiotypic mAb (Mitumomab - see, e.g., Chapman, Curr Opin Investig Drugs. 2003 Jun;4(6):710-5 and McCaffery et al., Clin Cancer Res. 1996 Apr;2(4):679-86), CeaVac® and related anti-idiotypic mAbs (see, e.g., Foon et al., J Clin Oncol. 1999 Sep;17(9):2889-5), anti- idiotypic mAb to MG7 mAb (see, e.g., Fengtian et al., Chin Med Sci J. 2002 Dec;17(4):215- 9), and other anti-cancer anti-idiotypic Abs (see, e.g., Birebent et al., Vaccine. 2003 Apr 2;21 (15):1601 -12, Li et al., Chin Med J (Engl). 2001 Sep;1 14(9):962-6, Schmitt et al., Hybridoma. 1994 Oct;13(5):389-96, Maloney et al., Hybridoma. 1985 Fall;4(3):191 -209, Raychardhuri et al., J Immunol. 1986 Sep 1 ;137(5):1743-9, Pohl et al., lnt J Cancer. 1992 Apr 1 ;50(6):958-67, Bohlen et al., Cytokines MoI Ther. 1996 Dec;2(4):231 -8, and Maruyama, J Immunol Methods. 2002 Jun 1 ;264(1 -2):121 -33). Such anti-idiotypic Abs can be advantageously optionally conjugated to a carrier, which may be a synthetic (typically inert) molecule carrier, a protein (e.g., keyhole limpet hemocyanin (KLH) (see, e.g., Ochi et al., Eur J Immunol. 1987 Nov;17(1 1 ):1645-8)), or a cell (e.g., a red blood cell - see, e.g., Wi et al., J Immunol Methods. 1989 Sep 1 ;122(2):227-34)). Compositions and combination administration methods of the invention also include the inclusion or coadministration of nucleic acid vaccines, such as naked DNA vaccines encoding such cancer antigens/tumor-associated antigens (see, e.g., US Patents 5,589,466, 5,593,972, 5,703,057, 5,879,687, 6,235,523, and 6,387,888).
In another aspect, the combination administration method and/or combination com- position comprises an autologous vaccine composition. In a further aspect, the combination composition and/or combination administration method comprises a whole cell vaccine or cytokine-expressing cell (e.g., a recombinant IL-2 expressing fibroblast, recombinant cyto- kine-expressing dendritic cell, and the like) (see, e.g., Kowalczyk et al., Acta Biochim Pol. 2003;50(3):613-24; Reilly et al., Methods MoI Med. 2002;69:233-57; and Tirapu et al., Curr Gene Ther. 2002 Feb;2(1 ):79-89). Another example of a therapeutic autologous cell method that can be useful in combination methods of this invention is the MyVax® Personalized Immunotherapy method (previously referred to as GTOP-99) (available through Genitope Corporation - Redwood City, CA, USA) (see US Patents 5,972,334 and 5,776,746).
In a further aspect, combination compositions and/or combination administration methods of the invention comprise administration of an immunomodulatory compound or modulator thereof (e.g., an anti-inhibitory immunomodulatory antibody). Examples of such compounds include T cell activating and proliferation-promoting molecules, such as B7 molecules (B7-1 , B7-2, variants thereof, and fragments thereof) (see, e.g., Adv Exp Med Biol. 2000;465:381 -90 and US Patent Application 20030208058), ICOS (inducible co- stimulator) molecules, and OX40 molecules (see Coyle et al., Springer Semin Immunopathol. 2004 Feb;25(3-4):349-59 and 6,312,700). Another example of such a molecule is an inhibitor of a negative T cell regulator, such as an antibody against CTLA4, such as MDX-010 (Phan et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100: 8372). Antibodies against several members of the TNF receptor (TNFR) family have been shown to augment T cell proliferative responses. Antibodies to CD27 have also been shown to enhance T cell proliferation. The interaction of the integrin family member LFA-1 (lymphocyte function-associated antigen 1 , or CD18/CD1 1 a) with its ligands intercellular adhesion molecule (ICAM)-I , -2 and -3 is well known to be an important participant in the activation of T cells. SLAM (signaling lymphocyte activation molecule, or CDw150) is another T cell regulator. The heat-stable antigen (HSA or CD24) is a glycophosphatidylinositol (GPI)-linked protein of 27 amino acids found on the surface of hematopoietic and neuronal cells in an extensively glycosylated 38-70 kDa form that enhances T cell proliferation. 4-1 BB is a co-stimulatory receptor for T cells. TNFR- associated factors (TRAFs) also are T cell signaling molecules. CD40L also can act as an immunomodulator. The CD2-LFA3 pathway also is important to T cell regulation (and ac- cordingly agents that act on it can be included in combination methods and compositions). NK cell activating and proliferating agents, such as stimulatory KIR molecules also can be included in such combination methods and compositions. Other immunomodulating agents that can also or alternatively be included in such combination compositions and methods are TGF-beta inhibitors. Cytokines and chemokines, which represent an important subset of immunomodula- tors, are discussed in detail in the following discussion. In one aspect, the invention provides combination compositions and methods comprising an antibody molecule and at least one anti-cancer cytokine, chemokine, or combination thereof. In general, any suitable anti-cancer cytokine and/or chemokine can be used with and/or combined with antibody molecules in the methods and compositions of this invention. Suitable chemokines and cytokines result in a detectably greater and/or more comprehensive immune response to cancer cells or related tissues (e.g., tumors) in vivo and do not substantially impede the binding of the α3 G1 G2(s) in the composition/method. Examples of suitable cytokines and growth factors include interferons (e.g., IFNβ,
IFNα (e.g., INFα2b), and IFNγ (e.g., IFNyI b)) and interleukins (e.g., IL-2, IL-4, IL-6, IL-7, IL- 10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, etc.). Additional cytokines that can be included in such compositions and methods include KGF, IFNβ, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFα (see, e.g., Dranoff, Nat Rev Can- cer. 2004 Jan;4(1 ):1 1 -22 and Szlosarek, Novartis Found Symp. 2004;256:227-37; discussion 237-40, 259-69).
Suitable chemokines can include Glu-Leu-Arg (ELR)-negative chemokines such as IP-10, MCP-3, MIG, and SDF-1 alpha from the human CXC and C-C chemokine families. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins (see, e.g., Eliason, BioDrugs, 2001 ;15(1 1 ):705-1 1 (with respect to PEGylated cytokines) and Shibuya et al., Laryngoscope. 2003 Nov;1 13(1 1 ):1870-84 (other cytokine derivatives), and WO 01/79258 (albumin-cytokine fusion proteins)).
These and other methods involving naturally occurring peptide-encoding nucleic acids herein can alternatively or additionally performed by "gene activation" and homologous recombination gene upregulation techniques (see, e.g., US Patents 5,968,502, 6,063,630, and 6,187,305 and European Patent Publication 0 505 500). Additionally useful cytokines for such combination therapies and compositions are described elsewhere herein.
In another aspect, the invention provides a combination composition or combination administration method comprising an antibody molecule and an adjuvant, typically in further combination with an anti-cancer immunogenic peptide (or surrogate nucleic acid/nucleic acid- encoding molecule). Examples of adjuvants include QS21 , GM-CSF, SRL-172, histamine dihydrochloride, thymocartin, Tio-TEPA, monophosphoryl-lipid A/micobacteria compositions, alum, incomplete Freund's Adjuvant, Montanide ISA, Ribi Adjuvant System, TiterMax adjuvant, syntex adjuvant formulations, immune-stimulating complexes (ISCOMs), GerbuR adju- vant, CpG oligodeoxynucleotides, lipopolysaccharide, and polyinosinic:polycytidylic acid. Combination compositions and combination administration methods also can involve "whole cell" and "adoptive" immunotherapy methods and "internal vaccination" techniques. For example, such methods can comprise infusion or re-infusion of immune system cells (e.g., tumor-infiltrating lymphocytes (TILs), such as CD4+ and/or CD8+ T cells (e.g., T cells expanded with tumor-specific antigens and/or genetic enhancements), antibody-expressing B cells or other antibody producing/presenting cells, dendritic cells (e.g., anti-cytokine expressing recombinant dendritic cells, dendritic cells cultured with a DC-expanding agent such as GM-CSF and/or Flt3-L, and/or tumor-associated antigen-loaded dendritic cells), anti-tumor NK cells, so-called hybrid cells, or combinations thereof (see, e.g., Fishman et al., Expert Rev Anticancer Ther. 2003 Dec;3(6):837-49; Whiteside et al., Cancer Immunol Immunother. 2004 Mar;53(3):240-8; Conrad et al., Curr Opin MoI Ther. 2003 Aug;5(4):405-12; Trefzer et al., MoI Biotechnol. 2003 Sep;25(1 ):63-9; Reinhard et al., Br J Cancer. 2002 May 20;86(10):1529-33; Korbelik et al., lnt J Cancer. 2001 Ju1 15;93(2):269-74; Costa et al., J Immunol. 2001 Aug 15;167(4):2379-87; Hanson et al., Immunity. 2000 Aug;13(2):265-76; Matsui et al., lnt Immunol. 2003 Jul;15(7):797-805; and Ho et al., Cancer Cell. 2003
May;3(5):431 -7). Cell lysates also may be useful in such methods and compositions. Cellular "vaccines" in clinical trials that may be useful in such aspects include Canvaxin™, APC- 8015 (Dendreon), HSPPC-96 (Antigenics), and Melacine® cell lysates. Antigens shed from cancer cells, and mixtures thereof (see, e.g., Bystryn et al., Clinical Cancer Research Vol. 7, 1882-1887, July 2001 ), optionally admixed with adjuvants such as alum, also can be advantageous components in such methods and methods. US Patent 6,699,483 provides another example of a whole cell anti-cancer therapy. Additional examples of such whole cell immunotherapies that can be usefully combined in antibody molecule-related compositions and methods are described elsewhere herein. In another aspect, one or more antibody molecules are delivered to a patient in association with the delivery of an effective amount of antigen-pulsed dendritic cells or other anti-cancer immune cells (e.g., NK cells).
In yet another aspect, an antibody molecule can be delivered to a patient in combination with the application of an internal vaccination method. Internal vaccination refers to induced tumor or cancer cell death, such as drug-induced or radiation-induced cell death of tumor cells, in a patient, that typically leads to elicitation of an immune response directed towards (i) the tumor cells as a whole or (ii) parts of the tumor cells including (a) secreted proteins, glycoproteins or other products, (b) membrane-associated proteins or glycoproteins or other components associated with or inserted in membranes, and/or (c) intracellular proteins or other intracellular components. An internal vaccination-induced immune response may be humoral (i.e. antibody - complement-mediated) or cell-mediated (e.g., the development and/or increase of endogenous cytotoxic T lymphocytes that recognize the internally killed tumor cells or parts thereof). In addition to radiotherapy, non-limiting examples of drugs and agents that induce tumor cell-death induction and internal vaccination methods include con- ventional chemotherapeutic agents, cell-cycle inhibitors, anti-angiogenesis drugs, monoclonal antibodies, apoptosis-inducing agents, and signal transduction inhibitors.
In another aspect, the invention provides combination compositions and combination administration methods that involve at least one antibody molecule and one or more cell cycle control/apoptosis regulators (or cell cycle/apoptosis "regulating agents"). A cell cycle control/apoptosis regulator that can be combined with antibody molecule^) can include, for example, one or more molecules that target and modulate cell cycle control/apoptosis regulators such as (i) cdc-25 (with NSC 663284 as a non-limiting example (see, e.g., Pu et al (2003) J Biol Chem 278, 46877)), (ii) cyclin-dependent kinases that over- stimulate the cell cycle (non-limiting examples of which are flavopiridol (L868275, HMR1275; Aventis), 7-hydroxystaurosporine (UCN-01 , KW-2401 ; Kyowa Hakko Kogyo), and roscovitine (R-roscovitine, CYC202; Cyclacel) - as reviewed by Fischer & Gianella-Borradori (2003) Exp Op Invest Drugs 12, 955-970), and (iii) telomerase modulators (such as BIBR1532 (Damm et al (2001 ) EMBO J 20, 6958-6968) and SOT-095 (Tauchi et al (2003) Oncogene 22, 5338- 5347)). Mycobacterium DNA has been reported to be capable of inducing apoptosis in can- cer cells (see, e.g., US Patent 6,794,368).
Non-limiting examples of molecules that interfere with apoptotic pathways include TNF-related apoptosis-inducing ligand (TRAIL)/apoptosis-2 ligand (Apo-2L), antibodies that activate TRAIL receptors, IFNs1 and anti-sense Bcl-2 (see lgney and Krammer (2002) Nature Rev. Cancer 2, 277-288; Makin; Dive (2003) Trends MoI Med 9, 2519; Smyth et al (2003) Immunity 18, 1 -6; and Panaretakis et al. (2003) Oncogene 22, 4543-4556).
In another aspect, the invention provides combination compositions and combination delivery methods comprising a telomerase inhibitor, telomerase vaccine, or combination thereof in addition to at least one antibody molecule or related molecule. Examples of such compositions and related techniques are described in US Patents 6,440,735 and 6,713,055. In yet another aspect, the invention provides combination compositions and combination administration methods that comprise one or more growth factor inhibitors.
A number of antibodies (e.g., mAbs) against growth factors and growth factor receptors are known that can be useful in promoting the treatment of cancer. For example, antibodies against the extracellular ligand binding domain of epidermal growth factor receptor (EGF-R) proteins that are abnormally activated in epithelial tumors can be useful in the treatment of aggressive epithelial cell-derived tumors. Antibodies against low molecular weight molecules and small molecules that inhibit the tyrosine kinase domains of such receptors also can be useful in combination compositions or combination administration methods. Non-limiting examples of such molecules include Herceptin (monoclonal antibody), Cetuxi- mab (monoclonal antibody), Tarceva (small molecule low molecular weight inhibitor), and lressa (small molecule low molecular weight inhibitor). Additional related and useful antibodies suitable for inclusion in such combination compositions and administration methods are described elsewhere herein.
In a further aspect, the invention provides combination compositions and methods that include one or more antibody molecules (or related molecule surrogates) and one or more inhibitors of angiogenesis, neovascularization, and/or other vascularization (such agents are referred to by terms such as anti-angoigenesis agents, anti-angiogenic drugs, etc. herein). Nonlimiting examples of such agents include (individually or in combination) en- dostatin and angiostatin (reviewed in Marx (2003) Science 301 , 452-454) and deriva- tives/analogues thereof; anti-angiogenic heparin derivatives and related molecules (e.g., heperinase III); VEGF-R kinase inhibitors and other anti-angiogenic tyrosine kinase inhibitors (e.g., SU01 1248 - see Rosen et al., Clinical Oncology; May 31 -June 3, 2003, Chicago, IL, USA (abstract 765)); temozolomide; Neovastat™ (Gingras et al., Invest New Drugs. 2004 Jan;22(1 ):17-26); Angiozyme™ (Weng et al., Curr Oncol Rep. 2001 Mar;3(2):141 -6); NK4 (Matsumoto et al., Cancer Sci. 2003 Apr;94(4):321 -7); macrophage migration inhibitory factor (MIF); cyclooxygenase-2 inhibitors; resveratrol (see, e.g., SaIa et al., Drugs Exp Clin Res. 2003;29(5-6):263-9); PTK787/ZK 222584 (see, e.g., Klem, Clin Colorectal Cancer. 2003 Nov;3(3):147-9 and Zips et al., Anticancer Res. 2003 Sep-Oct;23(5A):3869-76); anti- angiogenic soy isoflavones (e.g., Genistein - see, e.g., Sarkar and Li, Cancer Invest. 2003;21 (5):744-57); Oltipraz; thalidomide and thalidomide analogs (e.g., CC-5013 - see, e.g., Tohnya et al., Clin Prostate Cancer. 2004 Mar;2(4):241 -3); other endothelial cell inhibitors (e.g., Squalamine and 2-methoxyestradiol); fumagillin and analogs thereof; somatostatin analogues; pentosan polysulfate; tecogalan sodium; molecules that block matrix breakdown (such as suramin and analogs thereof (see, e.g., Marchetti et al., lnt J Cancer. 2003 Mar 20;104(2):167-74, Meyers et al., J Surg Res. 2000 Jun 15;91 (2):130-4, Kruger and Figg, Clin Cancer Res. 2001 Jul;7(7):1867-72, and Gradishar et al., Oncology. 2000 May;58(4):324- 33)); dalteparin (Scheinowitz et al., Cardiovasc Drugs Ther. 2002 Jul;16(4):303-9); matrix metalloproteinase inhibitors (such as BMS-275291 - see Rundhaug, Clin Cancer Res. 2003 Feb;9(2):551 -4; see generally, Coussens et al. Science 2002;295:2387-2392); angiocol; anti- PDGF mAbs and other PDGF (platelet derived growth factor) inhibitors; and PEDFs (pigment epithelium derived growth factors).
In another aspect, the invention provides combination compositions and combination administration methods wherein at least one antibody molecule is combined with or de- livered in association with a hormonal regulating agent, such as an anti-androgen and/or anti-estrogen therapy agent or regimen (see, e.g., Trachtenberg, Can J Urol. 1997 Jun;4(2 Supp 1 ):61 -64; Ho, J Cell Biochem. 2004 Feb 15;91 (3):491 -503), tamoxifen, a progestin, a luteinizing hormone-releasing hormone (or an analog thereof or other LHRH agonist), or an aromatase inhibitor (see, e.g., Dreicer et al., Cancer Invest. 1992;10(1 ):27-41 ). Steroids (of- ten dexamethasone) can inhibit tumour growth or the associated edema (brain tumors) and also can be suitable for combination with antibody molecules (or related compound surrogates thereof). One or more antibody molecules can be similar provided or combined with an antiandrogene such as Flutaminde/Eulexin; a progestin, such as hydroxyprogesterone caproate, Medroxyprogesterone/Provera, Megestrol acepate/Megace, etc.; an adrenocorti- costeroid such as hydrocortisone, prednisone, etc.; a luteinising hormone-releasing hormone (LHRH) analogue such as buserelin, goserelin, etc.; and/or a hormone inhibitor such as oc- treotide/Sandostatin, etc. In a particular aspect, antibody molecule(s) are provided or combined with an anti-cancer agent that is an estrogen receptor modulator (ERM) such as tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol/Estinyl, etc., or a combination of any thereof. Combination compositions and combination administration methods also or alternatively can comprise tamoxifen. Further teachings relevant to cancer immunotherapy are provided in, e.g., Berczi et al., "Combination Immunotherapy of Cancer" in NEUROIMMUNE BIOLOGY, Volume 1 : New foundation of Biology, Berczi I, Gorczynski R, Editors, Elsevier, 2001 ;pp.417-432. In a particular aspect, one or more antibody molecules (or suitable related molecule surrogates therefor - such substitution of antibody molecules with surrogates is contemplated throughout unless otherwise stated or clearly contradicted) is combined with or delivered in association with one or more aromatase inhibitors, such as anastrazole/Arimidex, aminoglutethimide/Cytraden, Exemestane, etc. Anti-aromatase agents inhibit the cyto- chrome P-450 component of the aromatase enzyme complex by interfering with the electron transfer from NADPH. Examples of such agents include anastrozole (Arimidex) and letrozole (Femara). These drugs can be also classified into first-generation (e.g. aminoglutethimide), second-generation (e.g. formestane and fadrazole) and third-generation (e.g. anastrozole, letrozole and exemestane) compounds. Anti-aromatase agents may also be divided into Type I and Type Il inhibitors. The Type I inhibitors have a steroidal structure similar to an- drogens and inactivate the enzyme irreversibly by blocking the substrate-binding site, and are therefore known as aromatase inactivators. Examples of such drugs include formestane and exemestane (Aromasin). Type Il inhibitors are non-steroidal and their action is reversible. Examples include anastrozole and letrozole. In one aspect, one or more antibody molecules are provided or combined with one or more of such molecules selected from Formestane, Exemestane, Aminoglutethimide, Anastrozole, and Letrozole.
Prostate cancer is often sensitive to finasteride, an agent that blocks the peripheral conversion of testosterone to 5-hydroxy-testosterone. Antibody molecules can be provided or combined with this agent or provided in association with various forms of androgen depri- vation therapy (ADT).
In one aspect, one or more antibody molecules are combined with or co-delivered with one or more intracellular signaling inhibitors. Examples of such compounds include tyrosine kinase inhibitors (Gleevec®, imatinib mesylate), modulators of the ras signaling pathway, and regulators of protein trafficking. Other examples include serine/threonine kinase inhibitors, protein-tyrosine phosphatases inhibitors, dual-specificity phosphatases inhibitors, and serine/threonine phosphatases inhibitors.
In another aspect, the invention provides combination compositions and combination delivery methods comprising one or more immune system inhibitors and one or more antibody molecules. Numerous immunosuppressive/immunomodulatory agents are known, examples of which include T lymphocyte homing modulators (e.g., FTY-720 - see, e.g.,
Yangawa et al., J Immunol. 1998 Jun 1 ;160(1 1 ):5493-9); calcineurin inhibitors (such as val- spodar, PSC 833, and other MDR-1 or p-glycoprotein inhibitors); and TOR-inhibitors (e.g., sirolimus, everolimus, and rapamcyin).
Other features the invention are combination compositions and combination delivery methods comprising one or more antibody molecules and one or more antineoplastic antibiotics. Such antibiotic chemotherapy agents prevent or delay cell replication. There are many differing antitumour antibiotics, but generally they prevent cell division by two ways: (1 ) binding to DNA making it unable to separate (2) inhibiting ribonucleic acid (RNA), preventing enzyme synthesis. Examples of such agents include Bleomycin (Blenoxane), Dactinomycin (Actinomycin D, Cosmegen), Daunorubicin (Cerubidine), Doxorubicin (Adriamycin, Rubex), ldarubicin (Idamycin), Mitomycin (Mitomycin-C, Mutamycin), Mitoxantrone (Novantrone), Pentostatin (Nipent), Plicamycin (Mithracin, Mithramycin), and combinations thereof.
In other aspects, an antibody molecule (e.g., an anti-G2 mAb) or related composition is delivered to a host in association with a thrombosis modulating agent such as a low molecular weight heparin, standard heparin, pentasaccharides, thrombin inhibitory agents (melagatran, ximelagatran, etc.), and/or coagulation factors like Factor VII, Factor VIII, etc.
Antibody molecules and pharmaceutical compositions of the invention are generally useful in the treatment of cancers associated with Ln5. As such, one aspect of the invention is embodied in a method of treating cancer by administering or otherwise delivering an effective amount of an antibody molecule to a mammalian host in order to promote, induce, or enhance a physiological effect associated with the treatment of cancer. Another facet of the invention is embodied in the use of an effective amount of one or more antibody molecules, having one or more of the above-described features, in the preparation of medicaments for the treatment of Ln5-associated cancers.
Unless otherwise stated or clearly contradicted by context, terms such as "treat", "treating", and "treatment" herein refer to the delivery of an effective amount of a therapeutically active compound or composition, such as antibody molecule or combination composition of the invention, with the purpose of preventing any symptoms or disease state to de- velop or with the purpose of easing, ameliorating, or eradicating (curing) such symptoms or disease states already developed. The term "treatment" is thus meant to include prophylactic treatment. However, it will be understood that therapeutic regimens and prophylactic regimens also can be considered separate and independent aspects of the invention (e.g., such regimens may differ in terms of dosage, dosage regimen, etc.). In practicing such methods and, accordingly, in preparing such medicaments, antibody molecules, related compounds (e.g., antibody molecule-encoding nucleic acids), and related compositions can generally be delivered (incorporated) in any suitable dosage. Determination of precise dosage for optimal effect will vary with a number of factors (condition to be treated, age of patient, health of patient, type(s) of antibody molecule or surrogate used, presence of additional active agents and/or application of related therapies, etc.), such that it is often more useful to describe dosage in terms of an amount sufficient or optimal for inducing, promoting, and/or enhancing a particular effect. In this respect, compositions of the invention can include "therapeutically effective amount," a "prophylactically effective amount", or "physiologically effective amount" of an antibody molecule (or "related composition" - e.g., an antibody-encoding nucleic acid, vector, host cell, etc.) or combination composition.
Drugs employed in cancer therapy may have a cytotoxic or cytostatic effect on cancer cells, or may reduce proliferation of the malignant cells. Among the texts providing guidance for cancer therapy is Cancer, PRINCIPLES AND PRACTICE OF ONCOLOGY, 4th Edition, De- Vita et al., Eds. J. B. Lippincott Co., Philadelphia, Pa. (1993). An appropriate therapeutic ap- proach is chosen according to such factors as the particular type of cancer and the general condition of the patient, as is recognized in the pertinent field.
In another aspect, the invention relates to a method of inducing, promoting, and/or enhancing one or more physiological events/activities in a patient (e.g., the reduction of can- cer progression). A "physiologically effective" amount refers to an amount that is sufficient to induce, promote, and/or enhance the desired physiological effect(s).
The compositions of the invention generally can be administered in any suitable dosage regimen. Suitability with respect to dosage regimens refers to the administration of any number of doses of a composition, any number of times in a relevant period (typically a day) that result in a desired physiological effect. Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It can be especially advantageous to formulate parenteral com- positions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the antibody molecule, related composition, or combination to be administered to the host, (b) the host, and (c) the particular therapeutic or prophylactic effect to be achieved. The total time of a course of treatment also can be any suitable time and also is likely to vary with a number of similar factors that will be determinable to skilled practitioners with routine experimentation. Dosage of anti-cancer agents, such as antibody molecules, typically is adjusted for the patient's body surface area (BSA), a composite measure of weight and height that mathematically approximates the body volume. Thus, in one aspect, the invention relates to the delivery of a BSA-adjusted amount of an antibody molecule or antibody molecule- comprising composition. The BSA is usually calculated with a mathematical formula or a nomogram, rather than by direct measurement. Such methods are known in the art.
An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody portion of the invention is about 0.1 -100 mg/kg, such as about 0.1 -50 mg/kg, for example about 0.1 -20 mg/kg, and more particularly about 1 -10 mg/kg (e.g., at about 0.5 mg/kg (such as 0.3 mg/kg), about 1 mg/kg, or about 3 mg/kg). Generally, such an amount is administered once per day or less (e.g., 2-3 times per week, 1 times per week, or 1 time every two weeks).
In the case of combination compositions, the antibody molecule or related compound is coformulated with and/or coadministered with one or more additional therapeutic agents as described elsewhere herein (e.g., an antigenic peptide or an immunostimulatory cytokine). Such combination therapies may require lower dosages of the antibody molecule and/or the co-administered agents, thus avoiding possible toxicities or complications associated with the various monotherapies.
Another aspect of the present invention provides a kit comprising an antibody mole- cule, related composition, or combination thereof, pharmaceutically excipient component, and optionally other pharmaceutical composition components (e.g., one or more secondary active agents). A kit may include, in addition to one or more antibody molecules, various diagnostic or therapeutic agents. A kit may also include instructions for use in a diagnostic or therapeutic method. Such instructions can be, for example, provided on a device included in the kit. Advantageously, such a kit includes an antibody molecule and a diagnostic agent that can be used in one or more of the various diagnostic methods described elsewhere herein. In another preferred embodiment, the kit includes an antibody molecule, related compound, or combination composition in a highly stable form (such as in a lyophilized form) in combination with pharmaceutically acceptable carrier(s) that can be mixed with the highly stable composition to form an injectable composition for near term administration.
Antibody molecules, related compounds, and combination compositions described above and elsewhere herein, such as the above-described pharmaceutically acceptable compositions, are useful in a variety of therapeutic and prophylactic regimens and diagnostic and prognostic applications. In one aspect, the invention provides a method of detection, diagnosis, prognosis, monitoring, and therapeutic treatment of cancer and aspects thereof (e.g., the invention provides a method of inhibiting the migration of invasive phenotype cancer cells through the delivery of an antibody molecule to the cancer cells or the vicinity of such cells, such as near the invasive front of a cancer cell population). In a particular exemplary aspect, the invention provides a method of preventing, delaying, minimizing, and/or reducing "cancer progression" in a mammalian host, such as a human patient, having a detectable level of cancer cells comprising administering an antibody molecule, a related composition (e.g., a nucleic acid encoding an antibody molecule, an anti-anti-idiotypic α3 G1 G2 antibody, etc.), or a combination composition of the invention an amount sufficient to detectably reduce the progression of the cancer in the host. In another aspect, the invention provides a method for the reduction or cessation of tumor progression in a mammalian host (e.g., a human), having a detectable level of cancer cells or pre-cancer cells comprising administering an antibody molecule, a pharmaceutical composition, or a related composition (e.g., a nucleic acid encoding an antibody molecule), in an amount sufficient to detectably reduce the progression of the cancer in the host.
Cancer cells are cells that divide and reproduce abnormally with uncontrolled growth (e.g., by exceeding the "Hayflick limit" of normal cell growth (as described in, e.g., Hayflick, Exp. Cell Res., 37,614 (1965)). "Cancers" generally consist of single or several clones of cells that are capable of partially independent growth in a host (e.g., a benign tumor) or fully independent growth in a host (malignant cancer). Cancer cells arise from host cells via neoplastic transformation ("carcinogenesis").
Terms such as "preneoplastic," "premalignant," and "precancerous" with respect to the description of cells and/or tissues herein refer to cells or tissues having a genetic and/or phenotypic profile that signifies a significant potential of becoming cancerous. Usually such cells can be characterized by one or more differences from their nearest non-neoplastic counterparts that signal the onset of cancer progression or significant risk for the start of cancer progression. Such precancerous changes, if detectable, can usually be treated with excellent results. In general, a precancerous state will be associated with the incidence of neoplasm(s) or preneoplastic lesion(s). Examples of known and likely preneoplastic tissues include ductal carcinoma in situ (DCIS) growths in breast cancer, cervical intra-epithelial neoplasia (CIN) in cervical cancer, adenomatous polyps of colon in colorectal cancers, atypical adenomatous hyperplasia in lung cancers, and actinic keratosis (AK) in skin cancers. Preneoplastic phenotypes and genotypes for various cancers, and methods for assessing the existence of a preneoplastic state in cells, have been characterized. See, e.g., Medina, J Mammary Gland Biol Neoplasia. 2000 Oct;5(4):393-407; Krishnamurthy et al., Adv Anat Pathol. 2002 May;9(3):185-97; Ponten, Eur J Cancer. 2001 Oct;37 Suppl 8:S97-1 13; Nik- linski et al., Eur J Cancer Prev. 2001 Jun;10(3):213-26; Walch et al., Pathobiology. 2000 Jan- Feb;68(1 ):9-17; and Busch, Cancer Surv. 1998;32:149-79. Gene expression profiles can increasingly be used to differentiate between normal, precancerous, and cancer cells. For example, familial adenomatous polyposis genes prompt close surveillance for colon cancer; mutated p53 tumor-suppressor gene flags cells that are likely to develop into aggressive cancers; osteopontin expression levels are elevated in premalignant cells, and increased te- lomerase activity also can be a marker of a precancerous condition (e.g., in cancers of the bladder and lung). In one aspect, the invention relates to the treatment of precancerous cells. In another aspect, the invention relates to the preparation of medicaments for treatment of precancerous cells.
"Cancer progression" refers to any event or combination of events that promote, or which are indicative of, the transition of a normal, non-neoplastic cell to a cancerous, neo- plastic cell, the migration of such neoplastic cells, and the formation and growth of tumors therefrom (which latter aspect can be referred to as tumor progression). Examples of such events include phenotypic cellular changes associated with the transformation of a normal, non-neoplastic cell to a recognized pre-neoplastic phenotype, and cellular phenotypic changes that indicate transformation of a pre-neoplastic cell to a neoplastic cell. Typical and specific stages of cancer include cell crisis, immortalization and/or normal apoptotic failure, proliferation of immortalized and/or pre-neoplastic cells, transformation (i.e., changes which allow the immortalized cell to exhibit anchorage-independent, serum-independent and/or growth- factor independent, or contact inhibition-independent growth, or that are associated with cancer-indicative shape changes, aneuploidy, and focus formation), proliferation of transformed cells, development of metastatic potential, migration and metastasis (e.g., the disassociation of the cell from a location and relocation to another site), new colony formation, tumor formation, tumor growth, and neotumorogenesis (formation of new tumors at a location distinguishable and not in contact with the source of the transformed cell(s)). Carcinogenesis, the initial stage of cancer progression, is typically associated with the activation of genes that regulate cell growth via bypassing the host cell's regulatory controls (e.g., bypassing or overcoming a host cell's normally active apoptotic signaling pathway(s)) and the reduced expression of tumor-suppressor genes. Neoplastic conversion is the transformation of a preneoplastic cell into one that expresses a neoplastic phenotype. Cancer progression often is also or alternatively (and more generally) described by the general stages of initia- tion, promotion, and progression. In tumor-forming cancers, for example, cancer progression often is described in terms of tumor initiation, tumor promotion, malignant conversion, and tumor progression (see, e.g., CANCER MEDICINE, 5th Edition (2000) B.C. Decker Inc., Hamilton, Ontario, Canada (Blast et al. eds.)). In another and later stage of cancer progression, immunogenic tumors typically escape immune-surveillance of the host enabling their growth. Additional mid to late stage aspects of cancer progression include evasion of apoptosis by the cancer cell, achieving limitless replication potential, achieving self-sufficiency in growth factor expression, achieving abnormal insensitivity to anti-growth signals; achieving sustained angiogenesis, and metastasis.
Metastasis refers to the stage of cancer progression associated with the spread of cancer cells from one site in a medium to another, such as in the tissue(s) of a patient. Me- tastasis also typically is involved with a number of distinct physiological events, which include the escape of cancer cells from an initial site via lymphatic channels or protease activity; the survival of cancer cells in circulation; arrest in secondary site(s); extravasation into surrounding tissue; initiation and maintenance of growth, and vascularization of metastatic tumor(s). Metastasis also may involve the ability of tumor cells to secrete proteases that allow invasion beyond the immediate location of the primary tumor. A prominent characteristic of malignant phenotype is the propensity for genomic instability and uncontrolled growth.
Metastatic cancer cells typically penetrate the extracellular matrix (ECM) and the basement membrane of the blood vessels to metastasize to a target organ (ectopic site). The EMC consists of proteins embedded in a carbohydrate complex (heparan sulfate peptidogly- cans), and proteases surrounding a tumor are active in this breaking down the host tissue. Thus, the penetration of the ECM and basement membranes and breakdown of related host tissues also are relevant aspects of cancer progression. Indeed, there often is a complex mix of the normally consecutive processes of cell attachment, detachment, as well as degra- dation of extracellular matrix proteins, and migration, which is needed for the locomotion of invasive tumor cells to distant locations. All of these activities are important aspects of cancer progression in the context of the present invention. Thus, for example, delivery of an antibody molecule, related compound, or combination composition can be used as a means of reducing any one of these physiological activities in association with the treatment of cancer in a patient.
In another stage of cancer progression, immunogenic tumors typically escape immune-surveillance of the host enabling their growth. Additional related aspects of cancer progression include evasion of apoptosis by the cancer cell, achieving limitless replication potential, achieving self-sufficiency in growth factor expression, achieving abnormal insensi- tivity to anti-growth signals; achieving sustained angiogenesis, and metastasis.
In general, antibody molecules of the invention can be used to treat patients suffering from any stage of cancer progression (and to prepare medicaments for reduction, delay, or other treatment of cancer progression), however the treatment of patients in the later stages of cancer progression with antibody molecules and compositions of the invention is also an advantageous aspect of the invention.
Cancer progression (and thus the reduction thereof) can be detected by any variety of suitable methods. Methods for detecting cancers and cancer progression include (a) clinical examination (symptoms can include swelling, palpable lumps, enlarged lymph nodes, bleeding, visible skin lesions, and weight loss); (b) imaging (X-ray techniques, mammography, colonoscopy, computed tomography (CT and/or CAT) scanning, magnetic resonance imag- ing (MRI), etc.); (c) immunodiagnostic assays (e.g., detection of CEA, AFP, CA125, etc.); (d) antibody-mediated radioimaging; and (e) analyzing cellular/tissue immunohistochemistry. Other examples of suitable techniques for assessing a cancerous state and cancer progression include PCR and RT-PCR (e.g., of cancer cell associated genes or "markers"), biopsy, electron microscopy, positron emission tomography (PET), computed tomography, immu- noscintigraphy and other scintegraphic techniques, magnetic resonance imaging (MRI), karyotyping and other chromosomal analysis, immunoassay/immunocytochemical detection techniques (e.g., differential antibody recognition), histological and/or histopathologic assays (e.g., of cell membrane changes), cell kinetic studies and cell cycle analysis, ultrasound or other sonographic detection techniques, radiological detection techniques, flow cytometry, endoscopic visualization techniques, and physical examination techniques.
In general, delivering antibody molecules of the invention to a subject (either by direct administration or expression from a nucleic acid) and practicing the other methods of the invention can be used to reduce, treat, prevent, or otherwise ameliorate any suitable aspect of cancer progression in a subject.
A reduction of cancer progression can include, e.g., any detectable decrease in (1 ) the rate of normal cells transforming to neoplastic cells (or any aspect thereof), (2) the rate of proliferation of pre-neoplastic or neoplastic cells, (3) the number of cells exhibiting a preneoplastic and/or neoplastic phenotype, (4) the physical area of a cell media (e.g., a cell cul- ture, tissue, or organ (e.g., an organ in a mammalian host)) comprising pre-neoplastic and/or neoplastic cells, (5) the probability that normal cells and/or preneoplastic cells will transform to neoplastic cells, (6) the probability that cancer cells will progress to the next aspect of cancer progression (e.g., a reduction in metastatic potential), or (7) any combination thereof. Such changes can be detected using any of the above-described techniques or suitable counterparts thereof known in the art, which typically are applied at a suitable time prior to the administration of a therapeutic regimen so as to assess its effectiveness. Times and conditions for assaying whether a reduction in cancer potential has occurred will depend on several factors including the type of cancer, type and amount of antibody molecule, related composition, or combination composition being delivered to the host. The accomplishment of these goals by delivery of antibody molecules of the invention is another advantageous facet of this invention.
Other methods useful for diagnosing cancer progression include tumor grading and staging methods, such as the American Joint Commission on Cancer grading system, the National Program of Cancer Registries "General Staging" method (also known as Summary Staging, California Staging, and SEER Staging), and/or commonly used specialized grading systems (e.g., a high Gleason tumor grade score is indicative of an aggressive cancer in the context of prostate cancer; a TNM (Tumor, Nodes, Metastasis) Staging System often is useful in the context of colorectal cancer, and the Scarff-Bloom-Richardson system often is used in the context of breast cancer assessments). Further methods for identifying cancer and/or diagnosing cancer progression include cancer gene-related DNA methylation (see, e.g., Carmen et al., J. Natl. Cancer Inst., 93(22) (2001 )), DNA cytometry, mitosis assays (as to frequency, normalcy, or both), pleomorphism evaluations, the presence of autocrine stimulatory loop activity, tubule formation measurements, keritinization assays, intercellular bridge formation assays, epithelial pearl detection, aberrant hormone receptor expression or form production assays (e.g., Her2 overexpression assays), and other cancer-associated gene expression assays (e.g., PRL-3 protein tyrosine phosphatase gene expression assays). The reduction of cancer progression, as measured by any of the foregoing assays, by delivery of one or more antibody molecules, is another advantageous facet of the invention.
In particular aspects, the invention provides a method of treating a cancer graded as greater than TO (e.g., T1 , T2, or T3). In another aspect, the invention provides a method of preventing the progression of a patient's tumor load from T2 or T3 to T4. In another aspect, the invention relates to the treatment of cancer in a patient classified as having a T4 state. In another aspect, the invention also or alternatively relates to the treatment of a patient classified as having an M1 cancer. In a further facet, the invention relates to the treatment of a cancer that is staged as more progressed than a stage I cancer (e.g., a stage Il and/or stage III cancer). In a further aspect, the invention provides a method of reducing or halting progression of such stage Il and/or stage III cancers to stage IV cancers. In another facet, the invention provides a method of treating a cancer classified as a stage IV cancer.
The reduction of cancer cell migration and invasiveness are particularly advanta- geous aspects of the invention. Accordingly, the detection of a reduction in cancer progression in one or both of these physiological responses is particularly useful. Methods suitable for assessing these forms of cancer progression include Boyden and Transwell chamber assays (see, e.g., US 20020052307; Hujanen and Terranova (1985) Cancer Res. 45: 3517- 3521 ; and Pelletier, A.J., Kunicki, T. and Quaranta, V. (1996), J. Biol. Chem. 271 :364); ma- trigel migration assays (see, e.g., Zhang et al., Oncogene. 2004 Apr 15;23(17):3080-8 and Knutson et al., Molecular Biology of the Cell, 7: 383-396, 1996); integrin betai assays (see, e.g., Berry et al., Breast Cancer. 2003;10(3):214-9); beta-catenin and related molecule assays (e.g., E-cadherin and/or "slug" gene expression assays); radiographic assays (such as barium radiographic invasiveness assays); positron emission tomography assessments; magnetic resonance imaging (MRI) techniques (e.g., measurement of tumor diameter and/or volume); DNA cytometry; mammographic measurements; fluorescence in situ hybridization (FISH) analysis methods (e.g., using nucleic acid probes relevant for cancer gene expression, such as HER-2/neu probes); biopsy; angiogenesis assessments; and measuring relevant invasiveness-associated biological markers (including endogenous Ln5, and particularly forms of Ln5, Ln5 gene expression levels/patterns, Ln5 nucleic acid methylation, and Ln5 fragments/portions (such as portions of Ln5 α3, portions of Ln5 γ2, γ2/α3 heterodimers, and β3 fragments) associated with cancer progression); simultaneous measurements of serum sCEA and TIMP1 ; etc.). The characterization of invasive cells is well known in the art. A discussion of invasive cell characteristics and related principles can be found in, e.g., King RJB (1996) Cancer Biology (Addison Wesley Longman Ltd., Harlow Essex) and Liotta and Stetler-Stevenson (1991 ) - Cancer Res 51 :5054s-5059s.
The methods of the invention can be used to reduce the cancer progression of any suitable type of cancer. Forms of cancer that may be treated by the delivery or administration of antibody molecules, antibody molecule compositions, and combination compositions provided by the invention include squamous cell carcinoma, leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, Burketts lymphoma, acute or chronic myelogenous leukemias, promyelocytic leukemia, fibrosarcoma, rhabdomyoscarcoma; melanoma, seminoma, teratocarcinoma, neuroblastoma, glioma, astrocytoma, neuroblastoma, glioma, schwannomas; fibrosarcoma, rhabdomyoscaroma, osteosarcoma, melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer, and teratocarcinoma. Antibody molecules and related compositions also can be useful in the treatment of other carcinomas of the bladder, breast, colon, kidney, liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroid or skin. Antibody molecules of the invention (and related compositions) also may be useful in treatment of other hematopoietic tumors of lymphoid lineage, other hematopoietic tumors of myeloid lineage, other tumors of mesenchymal origin, other tumors of the central or peripheral nervous system, and/or other tumors of mesenchymal origin. Advantageously, the methods of the invention also may be useful in reducing cancer progression in prostate cancer cells, melanoma cells (e.g., cutaneous melanoma cells, ocular melanoma cells, and/or lymph node-associated melanoma cells), breast cancer cells, colon cancer cells, and lung cancer cells. The methods of the invention can be used to reduce cancer progression in both tumorigenic and non-tumorigenic cancers (e.g., non- tumor-forming hematopoietic cancers). The methods of the invention are particularly useful in the treatment of epithelial cancers (e.g., carcinomas) and/or colorectal cancers, breast cancers, lung cancers, vaginal cancers, cervical cancers, and/or squamous cell carcinomas (e.g., of the head and neck). Additional potential targets include sarcomas and lymphomas. Additional advantageous targets include solid tumors and/or disseminated tumors (e.g., myeloid and lymphoid tumors, which can be acute or chronic).
Methods of the invention also or alternatively are advantageous in the treatment of colorectal cancers, breast cancers, lung cancers, vaginal cancers, cervical cancers, and/or squamous cell carcinomas (e.g., of the head and neck). Additional potential targets for therapeutic uses of antibody molecules, related compounds, and related compositions include sarcomas and lymphomas. Advantageous targets also include solid tumors and/or disseminated tumors (e.g., myeloid and lymphoid tumors, which can be acute or chronic). In another exemplary aspect, the invention provides a method of increasing the ratio of quiescent to invasive neoplastic cells in a mammalian host comprising administering a therapeutically effective amount of an antibody molecule or related composition of the invention so as to increase the ratio of quiescent to invasive cells in the host.
In yet another aspect, the invention provides a method of reducing the invasive po- tential of a population of cancer cells in a mammalian host (e.g., in a human patient) comprising administering a physiologically effective amount of an antibody molecule (e.g., a therapeutically effective amount of an anti-α3 G1 G2 mAb, a variant thereof, a fragment of either thereof, or a derivative of any thereof), related compound, or combination composition so as to detectably reduce the invasive potential of the cancer cells. In still another aspect, the invention provides a method of reducing cell migration, reducing tumor growth, reducing neoplastic and/or pre-neoplastic cell division, or any combination thereof in a mammalian host (e.g., a human patient in need thereof) comprising administering a therapeutically effective amount of an antibody molecule (e.g., an antibody molecule), related compound, or combination composition of the invention to the host so as to achieve the desired outcome(s).
In a further aspect, the invention provides a method of promoting remission of a cancer in a mammalian host, such as a human patient, comprising administering a composition comprising an antibody molecule, such as an anti-α3 G1 G2 mAb, that competes with mAb 7B2 (e.g., relatively inhibits at a level of at least about 10% as determined by ELISA, such as inhibits binding by about 15% or more, about 20% or more, about 25% or more, etc.) to the host, so as to promote cancer remission in the host. In a particular aspect, the invention provides a method for treating a local recurrence of a cancer in a human patient. In another aspect, the invention provides a method of treating a distant recurrence of a cancer in a human patient. In an even further aspect, the invention provides a method for reducing the risk of developing a cancerous condition, reducing the time to onset of a cancerous condition, reducing the severity of a cancer diagnosed in the early stages, and/or reducing the affected area of a cancer upon development thereof in a mammalian host, comprising administering to a host a prophylactically effective amount of an antibody molecule, related compound, or combination composition of the invention so as to achieve the desired physiological effect(s).
In another aspect, the invention provides methods for inhibiting tumor growth and/or metastasis in an individual in need thereof, comprising contacting the tumor with an amount of a antibody molecule, related composition, or combination composition of the invention, so as to inhibit tumor growth and/or metastasis. Target tumors can include, but are not limited to, carcinomas. Such carcinomas include, but are not limited to squamous cell carcinomas (including but not limited to squamous cell carcinoma of skin, cervix, and vulva), gastric carcinomas, colon adenocarcinomas, colorectal carcinomas, and cervical carcinomas. Other carcinomas that can be treated by inventive methods described herein include ductal mam- mary carcinomas. Other common cancers that can be treated by inventive methods described herein include malignant melanomas.
Inhibiting tumor growth generally means causing a reduction in the amount of tumor growth that would occur in the absence of treatment and/or substantially complete cessation of detectable tumor growth, and includes decreases in tumor size and/or decrease in the rate of tumor growth. Inhibiting metastases means to reduce the amount of tumor metastasis that would occur in the absence of treatment, and includes a relative decrease in the number and/or size of metastases.
In still a different aspect, the inventive methods can provide means for eliciting, promoting, and/or enhancing an anti-tumor effect by slowing the growth, spread, or growth and spread of the front of a tumor into surrounding tissues, or the expected growth, spread, or growth and spread of a tumor. Tumor cell growth inhibition can be measured by any suitable standard and technique using, e.g., other methods described herein and/or inhibition assays such as are described in WO 89/06692.
An additional aspect of the invention is to provide a method for inhibiting or slowing the growth and/or spreading of a tumor into surrounding tissue by delivering to a patient in need thereof antibody molecule, related compound, or combination composition.
Additional features of the inventive methods include the reduction in the size and/or number of and/or prevention of the formation of tubular networks associated with Ln5. Another feature of the invention is a method of preventing poorly aggressive neoplastic cells from developing a vasculogenic phenotype. Such aspects can be combined with the general feature of reducing the invasive potential (aggressiveness) and/or metastatic potential of cancers by such methods. Reduction of neoplastic and/or preneoplastic cell migration, reduction of cell division, and/or reduction of cell migration by administration of compositions of the invention are additional physiological endpoints that can be achieved by application of inventive methods described herein.
In a further aspect, the invention provides a method of increasing the likelihood of survival over a relevant period in a human patient diagnosed with cancer. For example, the invention provides a method of increasing the likelihood of survival about six months, about nine months, about one year, about three years, or longer after treatment with an antibody molecule composition of the invention, as compared to not receiving treatment with the antibody molecule composition (survival rates can be determined by, e.g., studies on a population of similar patients, such as in the context of a clinical trial).
In another aspect, the invention provides a method for improving the quality of life of a cancer patient comprising administering to the patient a composition of the invention in an amount effective to improve the quality of life thereof. Methods for assessing patient quality of life in cancer treatment are well known in the art (see, e.g., Movass and Scott, Hematol Oncol Clin North Am. 2004 Feb;18(1 ):161 -86; Dunn et al., Aust N Z J Public Health. 2003;27(1 ):41 -53; Morton and Izzard, World J Surg. 2003 Jul;27(7):884-9; Okamato et al., Breast Cancer. 2003;10(3):204-13; Conroy et al., Expert Rev Anticancer Ther. 2003 Aug;3(4):493-504; List et al., Cancer Treat Res. 2003;1 14:331 -51 ; and Shimozuma et al., Breast Cancer. 2002;9(3):196-202).
In a further aspect, inventive methods described herein can be applied to significantly reduce the number of cancer cells in a vertebrate host, such that, for example, the total number and/or size of tumors are reduced. Such methods can be applied to treat any suitable type of tumor including chemoresistant tumors, solid tumors, and/or metastasized tumors. In a related sense, the invention provides a method for killing preneoplastic and/or neoplastic cells in a vertebrate, such as a human cancer patient.
In another facet, the invention provides a method of treating a condition associated with Ln5 activity in a patient in need of such treatment or at substantial risk of developing a disease, disorder, or condition wherein such treatment is beneficial. Exemplary prophylactic applications of antibody molecules, related compounds (e.g., anti-G1/G2 Ab-encoding nucleic acids), and related compositions include reducing the severity of an imminent cancer, reducing the spread of an imminent cancer, reducing the likelihood of developing cancer, reducing the effects of an imminent cancer, or a combination of any thereof, etc. In another aspect, the invention provides methods for inhibiting tumor growth and/or metastasis in an individual in need thereof, comprising contacting the tumor with an amount of an antibody molecule, related compound, or related composition (e.g., a combination composition) of the invention, so as to inhibit tumor growth and/or metastasis. The invention similarly relates to the use of antibody molecules or such related compounds or related compositions in the preparation of medicaments for inhibiting tumor growth and metastasis.
In one aspect, the method is applied to reduce the growth of tumor(s) that secrete detectable amounts of Ln5 or Ln5 subunit(s), such as an abnormally high level of a Ln5 (e.g., a Ln5 form associated with cancer), a β3/γ2 heterotrimer peptide, a γ2 monomer peptide, or combination thereof, as compared to non-neoplastic epithelial cells. The phrase "laminin-5 secreting tumor" refers to a tumor that expresses detectable amounts of Ln5 or a fragment thereof, unless otherwise stated (e.g., where the tumor produces an abnormally high amount of an Ln5 peptide or subunit). In these and other aspects, subjects treated by delivery of antibody molecules, related compounds, or related compositions typically are mammals and commonly is a human. Ln5-secreting tumors include, but are not limited to, carcinomas.
Ln5-secreting carcinomas include, but are not limited to squamous cell carcinomas (including but not limited to squamous cell carcinoma of skin, hypopharynx, cervix, and vulva), gastric carcinomas, colon adenocarcinomas, colorectal carcinomas, and cervical carcinomas. Other carcinomas that can be treated by inventive methods described herein include ductal mam- mary carcinomas. The invention provides methods of treating these types of cancers and relates to the use of antibody molecules, related compounds, and related compositions for the preparation of medicaments to treat such conditions. Other common cancers that can be treated by inventive methods described herein include malignant melanomas.
Inhibiting tumor growth generally means causing a reduction in the amount of tumor growth that would occur in the absence of treatment and/or substantially complete cessation of detectable tumor growth, and includes decreases in tumor size and/or decrease in the rate of tumor growth. Inhibiting metastases means to reduce the amount of tumor metastasis that would occur in the absence of treatment, and includes a relative decrease in the number and/or size of metastases. In another aspect, the invention provides a method for inhibiting migration of one or more laminin-5 secreting tumor(s), cancer cells/cancer cell populations, preneoplastic cells/cell populations, or population of other epithelial cells in a human patient comprising delivering an antibody molecule, such as an anti-α3G1 G2 antibody of the invention, a related compound (e.g., a DNA encoding an antibody molecule), or a related composition of the in- vention to the tumor(s), cell population(s), etc. or to an area sufficiently near the tumor(s), cell population(s), etc., and in an amount and under conditions such that migration of the Ln5 secreting tumor(s) or cell(s) is/are detectably inhibited (as determined by cessation of movement of the tumor in the patient or by comparison of tumor migration after administration of the antibody molecule against tumor migration at a similar stage in cancer progression in a suitably sized population of similar patients not treated with the antibody molecule, related compound, or related composition, such as may be determined through clinical trials involving administration of such compositions).
In one aspect, "migration" of cells means persistent migration (i.e., in such an aspect, the invention provides a method of reducing persistent migration in cancer cells). In another aspect, the invention provides a method of reducing neoplastic and/or preneoplastic cell polarization in a mammalian host, such as human, by delivery of an antibody molecule, related compound, or related composition of the invention.
In another aspect, such methods can be used to reduce the invasive/migratory potential of tumor cells. In still a different aspect, the inventive methods can provide means for eliciting, promoting, and/or enhancing an anti-tumor effect by slowing the growth, spread, or growth and spread of the front of a tumor into surrounding tissues, or the expected growth, spread, or growth and spread of a tumor. Tumor cell growth inhibition can be measured by any suitable standard and technique using, e.g., other methods described herein and/or inhibition assays such as are described in WO 89/06692. In a further aspect, inventive methods described herein provide a mechanism for detectably retarding the invasive/migratory potential of Ln5 positive cancer cells (e.g., by administering an anti-α3 G1 G2 mAb of the invention). The invention provides a mechanism for detectably retarding the invasive and/or migration potential of an Ln5 positive cancer cell by exposing said cell to a monoclonal antibody specific to the α3 chain of laminin-5. Inventive methods provided herein additionally can be applied to detectably disrupt contacts and/or associations between cancer cells, such as invading malignant cells, and an associated matrix, e.g., a provisional matrix of the type found in the immediate surroundings of such cells. The invention further provides a method for reducing the scattering of tumor cells in a mammalian cell population, such as a population of tumor cells in a human patient, an animal model, or in culture, comprising delivering an antibody molecule, a related compound, or a related composition (e.g., a combination composition) of the invention to the cells under conditions and in an amount such that tumor cell scattering is detectably reduced. Again, in this and other aspects of the invention, the term delivery means delivery by any suitable technique including, e.g., by expression from a gene transfer vector, by direct administration (e.g., injection in solution or biolistic delivery), or any other suitable method. Another feature of the invention is a method for inhibiting the formation of budding tumor cells and/or reducing tumor cell budding in a tumor comprising delivering an antibody molecule of the invention, related compound, or related composition of the invention in an amount and under conditions suitable for detectably inhibiting budding tumor cell formation and/or generally reducing tumor cell budding a tumor. Tumor cell budding in, e.g., colorectal carcinoma has been associated with the presence of intracellular laminin-5 (See, e.g., Sor- dat, et al., J. Pathol. 185: 44-52, 1998). As with other aspects of the invention directed to inhibition or reduction, such measurements can be made with respect to an individual, if appropriate (e.g., a detection that ongoing tumor cell budding has ceased in an individual would be one measure of the successful application of this method) and/or in the context of a patient population wherein successful application of the method is measured against the typical biological phenomena observed in a control group of a substantially similar pool of patients (e.g., patients suffering from a carcinoma associated with budding tumor cells).
The inventive methods also can be used to reduce the mimicry of embryonic vascu- logenesis by aggressive cancer cells. Vasculogenic mimicry is described in, e.g., Seftor et al., Cancer Res. 2001 Sep 1 ;61 (17):6322-7. Inventive methods provided here also or alternatively can be used to reduce the formation of neoplastic tubules and/or the dissociation of cells from neoplastic tubules.
An additional aspect of the invention is to provide a method for inhibiting or slowing the growth and/or spreading of a tumor into surrounding tissue by delivering to a patient in need thereof an anti-α3G1 G2 Ab molecule or other effective antibody molecule, related compound, or combination composition.
In another aspect, the invention provides a method for modulating (e.g., interfering, such as inhibiting) tumor cell-basement membrane interaction and/or adhesion comprising administering or otherwise delivering an antibody molecule of the invention, a related compound, or a combination composition in an amount effective to reduce tumor cell-basement membrane interaction and/or adhesion. Thus, in one aspect, inventive methods described herein can be applied to reduce tumor cell/basement membrane adhesion. Such adhesion is important, if not crucial, for the invasion of non-malignant tissues by epithelial cancer cells. Another feature of the inventive methods described herein is the ability to apply such methods to interfere with interactions between aberrant α3 chain peptides or fragments and surrounding tissues or cells.
Yet another feature of inventive methods provided herein is the ability to inhibit the conversion of carcinoma cell phenotype from epithelial to spindle-shaped (e.g., reduce the rate of such conversion, reduce the total number of converted cells, etc., with respect to the human patient and/or a population of substantially similar patients). Thus, in an exemplary aspect, the invention provides a method for reducing the conversion of such a phenotypic change which method comprises delivering an effective amount of an antibody molecule to precancerous epithelial cells so as to reduce such conversion. Inventive methods provided herein also can be particular advantageous in the context of inducing, promoting, and/or enhancing a physiological response associated with the treatment of cancer and/or reducing at least one aspect of cancer progression in cancer cells classified as poorly differentiated (see, e.g., Sordat et al., J. Pathol., 185:44-52 (1998) for an example of research discussing such poorly differentiated cancer cells). Inventive methods provided herein also can be particularly advantageous with respect to the elimination (partial or total) of micrometastases and/or for the prevention of a recurrence of cancer in a patient previously diagnosed with cancer but currently in a state of remission. Methods for assessing recurrence and/or the risk of recurrence are known in the art (see, e.g., US Patent 6,656,684) and can include application of other cancer diagnostic methods described herein. Additional aspects related to recurrence and remission are discussed elsewhere herein.
Delivering an antibody molecule, related compound, and/or related composition of the invention to a mammalian host, such as a human patient, also provides a method for reducing the migration of epithelial cells generally, including noncancerous epithelial cells (the role of Ln5 in epithelial cell migration is discussed in, e.g., Verrando, et al., Lab Invest. 71 : 567-74, 1994; Kikkawa, et al., J. Biochem. (Tokyo). 1 16: 862-9, 1994; Zhang, et al., Exp. Cell. Res. 227: 30922, 1996; OToole, et al., Exp. Cell. Res. 233: 330-9, 1997; Tani, et al., Am. J. Pathol. 151 : 1289-302, 1997; and SaIo, et al., Matrix Biology, in press, 1999). Ln5 is associated with epithelial cell migration in, for example, wound healing as well as cancer. For this and other reasons, the method of this aspect can be applied either to inhibit epithelial cell migration in both malignant and nonmalignant cells. Thus, for example, in one aspect, the invention relates to the use of antibody molecules, related compounds, and/or related compositions in the preparation of a medicament for modulating wound healing or for a method of modulating wound healing activities (e.g., in a patient in need thereof), for exam- pie by modulating (e.g., reducing) epithelial cell migration (e.g., keratinocyte migration) in the context of wound healing in a mammalian host.
In another aspect, inventive methods provided herein can be used as a technique for modulating cell differentiation and/or cell proliferation. As indicated above, inventive methods also can be used to reduce cell migration. Cell migration assays are described in US Patent Application No. 10/695,559. Thus, for example, the invention provides a method for blocking migration of epithelial cells comprising delivering to such cells an inhibitory amount of a composition comprising one or more antibodies against α3G1 G2.
A further aspect of the invention is to provide a method for promoting an immune response against a tumor by administering an antibody molecule, such as a monoclonal anti- body against α3 G1 G2, to a patient in need thereof in an amount and under conditions sufficient to induce a detectable immune response against the tumor. Inducement of an immune response can be measured by any suitable result, such as the inducement, promotion, and/or enhancement of Ab-mediated opsonization, complement-mediated response, etc. In a particular aspect, inventive methods described here provide a mechanism for inhibiting the formation of interactions between α3G1 G2 and other Ln5 binding molecules or Ln5 chains involving Cys442 comprising administering an antibody molecule, such as an anti-α3 G1 G2 mAb, which is specific for a portion of α3G1 G2 that comprises Cys442 or that is near enough to Cys442 to cause steric hindrance that prevents peptide binding to α3 G1 G2 at Cys442, such that access to Cys442 is effectively blocked by the binding of the an- tibody molecule.
In another exemplary aspect, the invention provides a method for reducing the adhesion between cancer cells and matrix structures such as the basement membrane. Cell adhesion assays for Ln5 associated cells are described in, e.g., US Patent Application 10/695,559. Moreover, the adhesion properties of laminin-5 have been demonstrated in several cell attachment studies (see, e.g., Carter et al., (1991 ) Cell 65: 599-610; Rousselle et al., (1991 ) J. Cell Biol. 1 14: 567:576; Sonnenberg et al., (1991 ) J. Cell Biol. 1 13: 907-917; Niessen, et al., (1994) Exp. Cell. Res. 21 1 : 360-367; and Rousselle et al. (1994) J. Cell Biol. 125:205214)). Thus, in an exemplary aspect, the invention provides a method of reducing cancer cell-matrix adhesion comprising administering an effective amount of an antibody molecule to a mammalian host so as to detectably reduce such adhesion.
The adhesive function of laminin-5 has been shown to be mediated through, e.g., α3β1 and α6β4 integrins (see, e.g., Carter et al. (1991 ) Cell 65: 599-610; Sonnenberg (1991 ) J. Cell Biol. 1 13: 907-917; and Rousselle (1994) J. Cell Biol. 125: 205214)). Thus, in one variation of the aspect of the preceding paragraph, an antibody molecule, such as an anti-α3 G1 G2 antibody, can be administered with a molecule that targets and binds α3β1 integrin, α6β4 integrin, α6β1 integrin, or combination of any thereof, such as antibodies specific for these integrins, thereby further blocking Ln5-mediated association of cells and the surrounding matrix, such as the ECM or lamina lucida. In another similar aspect, the invention provides a method of reducing tumor cell invasiveness in a mammal, such as a human patient, comprising delivering an antibody molecule to the mammal and a protein that binds (and desirably inhibits) a matrix metallopro- teinase-2 (MMP-2), such as an anti-MMP2, under conditions and in respective amounts such that tumor cell invasiveness is reduced (surrogate nucleic acid(s) also can be used in such a method). The invention also provides combination compositions characterized by inclusion of effective amounts of such molecules. The role of Ln5 and MMP-2 in tumor cell budding and invasiveness is explored in, e.g., Masaki et al., Anticancer Res. 2003 Sep- Oct;23(5b):41 13-9. In this and other suitable combination aspects of the invention, a bispeci- fie antibody comprising a set of VH and VL sequences specific for α3G1 G2 and another set of VL and VL sequences specific for a second molecule, such as MMP-2, can be used as an alternative to delivery of two separate molecules (bispecific antibodies and combination compositions that comprise similar combinations that may likewise be suitable basis for mul- tispecific antibodies are discussed elsewhere herein). In this aspect and other combination administration method aspects, a single administration of a combination compound of the invention can be employed in place of a separate delivery strategy (here, e.g., a composition comprising an anti-α3G1 G2 antibody and a anti-MMP-2 antibody can be used to so inhibit cell invasiveness, rather than requiring both antibodies to be administered to a host).
In another sense, the invention provides a method of increasing the likelihood of survival over a relevant period in a human patient diagnosed with cancer. For example, the invention provides a method of increasing the likelihood of survival about six months, about nine months, about one year, about three years, about five years, about seven years, about ten years, or more, after treatment with an antibody molecule or antibody molecule composition of the invention, as compared to not receiving treatment with the antibody molecule or related composition (survival rates can be determined by studies on a population of similar patients, such as in the context of a clinical trial).
In another aspect, the invention provides a method for improving the quality of life of a cancer patient comprising administering to the patient a composition of the invention in an amount effective to improve the quality of life thereof. Methods for assessing patient quality of life in cancer treatment are well known in the art (see, e.g., Movass and Scott, Hematol Oncol Clin North Am. 2004 Feb;18(1 ):161 -86; Dunn et al., Aust N Z J Public Health. 2003;27(1 ):41 -53; Morton and Izzard, World J Surg. 2003 Jul;27(7):884-9; Okamato et al., Breast Cancer. 2003;10(3):204-13; Conroy et al., Expert Rev Anticancer Ther. 2003 Aug;3(4):493-504; List et al., Cancer Treat Res. 2003;1 14:331 -51 ; and Shimozuma et al., Breast Cancer. 2002;9(3):196-202). In a more particular aspect, any one of the methods described herein, such as the reduction of invasive cancer cells, is practiced by a method that comprises delivering an antibody molecule to a tissue that is characterized by a lack of mature hemidesmosomes as compared to healthy (nonmalignant) basement membrane-associated tissues. In this and other aspects, the delivery of an antibody molecule can be considered to provide a mechanism for modulating the architecture of the basement membrane in a mammalian host, such as a human cancer patient (e.g., for increasing the amount of mature hemidesmosome- containing tissue in a patient). However, in another aspect, administration of an antibody molecule can provide a mechanism for inhibiting hemidesmosome assembly. In a further aspect, the inventive methods described herein also or alternatively provide a method for impeding branching morphogenesis of epithelial and epithelial-derived cells. In still another aspect, such methods can modulate epithelial cell polarization.
Inventive methods also can be used as a means to interfere with Ln5 interaction with interacting molecules (examples of which are discussed elsewhere herein), such as Ln5 interacting integrins (e.g., administration of an antibody molecule can be used to interfere with Ln5:α6β1 integrin interactions).
In a further aspect, the inventive methods can be used to decrease the rate of an- giogenesis and/or neovascularization, such as ePTFE-associated neovasculariza- tion/angiogenesis, in a host, such as in a human cancer patient. In yet another aspect, the inventive methods can be used to delay, reduce, and/or prevent the loss of normal basement membrane barrier structures in the course of cancer progression and/or angiogene- sis/neovascularization.
In a further aspect, the invention provides a method of modulating a Ln5-associated signaling pathway(s), such as in the context of regulating apoptosis in a cancer cell, compris- ing delivering an antibody molecule to the cell population in an amount and under conditions such that oc3 G1 G2 is bound by the antibody molecule in a manner resulting in modulation of a Ln5-associated signaling pathway. For example, such methods can be used to modulate RAC and/or NFKB activation, so as to, for example, impede RAC-mediated sustenance of tumor cell viability. In yet another facet, various methods of the invention can be used as a method of modulating Ln5 associated protein kinase C (PKC), phosphoinositide 3-OH kinase (PI3-K), Akt, and/or MAP kinase activities/pathway(s). In another aspect, the invention provides combination compositions and methods involving inhibitors of one or more of these pathways. In a specific example, the invention provides combination compositions and methods that involve at least one antibody molecule and LY294002 and/or Wortmannin (these compounds and their use are known in the art - see, e.g., Fukuchi et al., Biochim Bio- phys Acta. 2000 Apr 17;1496(2-3):207-20; Yu et al., MoI Carcinog. 2004 Oct;41 (2):85).
In a further aspect, inventive methods taught herein can be used to reduce hematogenous metastasis and/or tumor cell arrest (e.g., in the context of pulmonary metastases) in a human patient.
Antibody molecules, related molecules, and/or related compositions (e.g., combination compositions) of the invention can be administered to achieve any combination of the aforementioned physiological responses and promote any of the above-described therapeutic and/or prophylactic regimens. Thus, for example, in one aspect the invention provides a method of reducing the migration and invasiveness of epithelial-derived cancer cells in a human patient in need thereof by, among other things, delivering an amount of an antibody molecule, a combination composition (any of which such compositions may be delivered by, e.g., expression of multiple nucleic acid sequences encoding a αG1 G2 and other anti-cancer peptide(s)), an antibody molecule related compound, or a combination thereof, such that mi- gration and invasiveness of epithelial-derived cancer cells is detectably reduced.
In another aspect, the invention provides a method of reducing the risk of a start of cancer progression, reducing the risk of further cancer progression in a cell population that has undergone initiation, and/or providing a therapeutic regimen for reducing cancer progression in a human patient, which comprises administered an amount of an antibody mole- cule, a related compound, or combination composition (or applying a combination administration method) to a patient that has been diagnosed as having cells exhibiting preneoplastic and/or neoplastic cell-like levels and/or types of gene expression, such as cancer-associated patterns of erbB2 (Her2/neu) gene expression; p53 gene expression; BRCA1 and/or BRCA2 gene expression; PTEN gene expression; ras family gene expression (k-ras, h-ras, m-ras, RAB2, RAP2A, etc.), c-MYC gene expression or cancer-like expression of one or more of the following Exol, ASPP2, C/EBPD, p16(INK4a) CDKN2A, R24P, P81 L, V126D, BNIP3, MYH, PTCH, B-ras, A-ras, PPAR (α, Y, and Δ), MC1 R, TP16p14/ARF, SMAD3, SMAD4, CDK4, p73, p15, AXIN1 , raf, CHEK2, SHIP, HFE, p21 (CIP1/WAF1 ), FAS, TSG101 , MEN1 , GSTPI, P2X7, BRAF, HPV type 16 E7, P27 Cyclin E, Cyclin D, Rb, P300, Mdm2, Fos, Jun, N-Ras, Ki-Ras, RaM , AbI, Bcl-2, Bcl-6, Bax, APC (Accession No: M74088), Beta catenin, E- cadherin, PI3-kinase, TGFα, TGFβ, TGFβ receptor, Src, Met, Akt, AIk, Grb2, She, and E2F 1 -5. In a particular exemplary aspect, the invention provides a method of inhibiting cancer progression (either before or after detection of any aspect thereof) in a human exhibiting cancer-like upregulation/expression of Ras and Myc; expression of Ras with loss of regular p53 gene activity; expression of Ras with loss of regular Rb activity; expression of Ras with loss of regular NFKβ activity; Expression of Ras with loss of regular APC activity; expression of Ras with loss of regular Arf activity; expression of Ras with E7; etc.
Additional benefits of inventive methods include the reduction or prevention of cancer progression in a pre-invasive lesion or growth; inducing, promoting, and/or enhancing tumor regression; enhancing a patient's immune system attach against cancer tumors; enhancement of the effectiveness of an anti-cancer agent; treating a pre-neoplastic or neoplastic disease characterized by abnormal expression of Ln5 and/or producing promigratory α3- associated peptides; reducing cancer progression in cell exhibiting marked nuclear atypia; and treating a human for a condition associated with an undesirable epithelial and/or epithe- lial-derived cell proliferation.
In one aspect, an antibody molecule (or related compound surrogate) is administered or otherwise delivered, in an effective amount, to or near an intraepithelial neopla- sia/lesion so as to reduce cancer progression therein, for example in a squamous intraepithelial lesion. In another aspect, inventive methods described herein provide a method of treating cancer in premalignant phase/preinvasive phase cells.
In a further aspect, inventive methods described herein can be applied to significantly reduce the number of cancer cells in a vertebrate host, such that, for example, the total number and/or size of tumors is/are reduced. Such methods can be applied to treat any suitable type of tumor including chemoresistant tumors, solid tumors, and/or metastasized tumors. In a related sense, the invention provides a method for killing preneoplastic and/or neoplastic cells in a vertebrate, such as a human cancer patient.
Inventive methods provided herein can be used to reduce the number, spread, and/or development of metastases in a chordate, such as in a human cancer patient. In a particular aspect, the invention provides a method of reducing the number of metastases in the tissue(s) of a chordate host, such as a mammal, for example a human patient, by at least about 30%, such as about 40% or more, about 50% or more, about 60% or more, or about 70% or more (e.g., about 35-75%) as compared to non-treatment in a substantially similar host over a period of about 3 or more days, such as about one week or more (e.g., about 2, 3, 4, 6, 8, 10, or 12 weeks; about 4, 5, 6, 9, or 12 months; or longer). Another advantageous aspect of the invention is the reduction of cancer progression in a patient having a cancer staged by the presence of a significant number of poorly differentiated and/or undifferentiated cancer cells (highly anaplastic cells). Antibody molecules, such as conjugated α3 G1 G2 s, may be useful in reducing cancer progressions in such patients where other methods have been ineffective. In another aspect, the invention provides a method wherein an antibody molecule is used to target other molecules to migrating cells, such as migrating and invasive cancer cells, or to other molecules, cells, and/or tissues associated with α3-associated proteins.
In one such aspect, an antibody molecule is a conjugated molecule, as described elsewhere herein, wherein, for example, the conjugate comprises a radioisotope, a toxin, an apoptotic domain/peptide, or other cytotoxic and/or anti-cancer agent. Such methods are especially useful in targeting the invasion front of a human carcinoma.
Several immunoconjugates, particularly those that incorporate internalizing antibodies and tumor-selective linkers, may exhibit impressive activity against cancer cells in pre- clinical models. Immunoconjugates that deliver doxorubicin, maytansine, and calicheamicin are examples of such molecules. Gemtuzumab ozogamicin, a calicheamicin conjugate that targets CD33, is an immunoconjugated molecule that has recently been approved by the US Food and Drug Administration (FDA) for treatment of acute myelogenous leukemia (AML). Such immunoconjugates are described in, e.g., Trail et al., Cancer Immunol Immunother. 2003 May;52(5):328-37 and Payne, Cancer Cell. 2003 Mar;3(3):207-12. A number of other conjugated molecules are described elsewhere herein as are combination partners that can also make conjugate partners and/or fusion partners in the context of antibody molecules to the extent such molecules are suitably able to bind α3 or an associated portion of Ln5.
In one aspect, the invention combines an antibody molecule that is conjugated to a molecule that can be used as a reporter or label, such as a green fluorescent protein (GFP) domain, or a radionuclide, and used to identify the location of tumors. In one exemplary aspect, the invention provides a method for identifying a tumor associated with an identified metastases or other identified cancer cell population in a human patient comprising administering such a composition and identifying locations where the antibody molecule has gath- ered, indicating the presence of migrating epithelial or epithelial-derived cells, such as invasive carcinoma cells, and/or the presence of α3-associated peptides (e.g., α3 -associated peptides secreted from cancerous or precancerous cells). Using such information, targeted application of radiation therapy, chemotherapy, or application of surgical techniques (e.g., a convention technique such as a biopsy, preferably a minimally invasive technique such as laser-assisted surgery, or an alternative surgical technique such as cryosurgery) can be used to efficiently reduce, isolate, or destroy a cancerous growth associated with Ln5 or a fragment thereof. Surgery in such contexts can include primary surgery for removing one or more tumors, secondary cytoreductive surgery, and palliative secondary surgery.
In another aspect, antibody molecules can be used in a pre-targeting protocol. In such protocols, primary molecules (here one or more antibody molecules) are allowed to bind targets (e.g., α3-associated peptides) and anti-antibody molecule antibody conjugates are thereafter used to deliver payload(s) to the vicinity of the targets.
In another aspect, the invention provides a method of delivering a prodrug to target cells for targeted therapy. Many therapeutic agents are administered as prodrugs. A prod- rug typically is a chemically modified form of the therapeutic agent designed to improve either its pharmacokinetic, pharmacological, or toxicological profiles. A prodrug is typically administered in a masked state. A chemical reaction, which typically is enzymatically facilitated, is usually required for prodrug activation.
In one aspect of the invention, antibody molecules are used to deliver a conjugated prodrug that can be activated by an exogenous enzyme that is subsequently or simultaneously administered to a patient. In another aspect, antibody molecules are used to deliver a conjugated enzyme that can be used to activate a subsequently or simultaneously delivered prodrug. Antibody-directed enzyme prodrug therapy (ADEPT) methods, such as described in the foregoing sentence, are known in the art (see, e.g., Bagshawe et al., Br. J. Cancer 58, 700-703, 1988; Senter et al., PNAS 85, 4842-4846, 1988; Niculescu-Duvaz, et al., Adv. Drug Delivery Rev. 26, 151 -172, 1997; and International Patent Application WO 93/02703).
Clinically useful prodrug activation systems, such as anti-cancer prodrug systems, can be delivered as masked antibody molecule-prodrug conjugates in combination with exogenous activating enzymes, by which the masked conjugated prodrugs are unmasked and thereby allowed to effect pharmacological effects, such as cytotoxic effects, on target cells. Prodrugs should typically not be activated by endogenous enzymes and are composed accordingly. Thus, for example, in ADEPT therapies bacterial enzymes may be used. In view of the immunogenicity of such enzymes, ADAPT therapy involving "human" or "humanized" catalytic antibody molecule antibodies can be more effective and/or less associated with negative side effects in therapeutic application.
In another aspect, a catalytic antibody molecule antibody is administered in association with a masked prodrug. Such catalytic antibodies typically are selected to catalyze the reaction that is not catalyzed by endogenous enzymes. Prodrug activation by catalytic antibodies (abzymes) specific for a target cell (here, an NK cell and/or another STM-associated cell, such as an NK target cell) is commonly referred to as antibody-directed abzyme prodrug therapy (ADAPT), ADAPT methods can be performed with catalytic antibody molecule antibodies and such antibodies are another feature of the invention. In one aspect, the invention provides a catalytic antibody molecule specific for both α3 and an enzymatic substrate site. Masked prodrugs can include prodrug versions of anti-cancer agents such as doxorubicin and camptothecin (see, e.g., Barbas et al., Proc. Natl. Acad. Sci USA 96, 6925- 6930, 1999), and phenolic N-mustards. Additional methods and principles relevant to such prodrug-associated therapies are provided in, e.g., United States Patents 5,807,688 and 6,268,488; Miyashita, et al., Proc. Natl. Acad. Sci. USA 90: 5337-5340 (1993); Campbell, et al., J. Am. Chem. Soc. 1 16: 2165-2166 (1994); Wentworth, et al., Proc. Natl. Acad. Sci. USA 93: 799-803 (1996); and Kakinuma et al., J Immunol Methods. 2002 Nov 1 ;269(1 -2):269-81 . As indicated already herein, antibody molecules can be delivered in association or combination with one or more active agents or therapeutic methods. In this respect, generally any combination composition described herein is to be understood as providing support for co-delivery methods involving the various secondary compounds. In one aspect, the invention provides a method of delivering one or more antibody molecules in association with application of anti-cancer chemotherapy and/or radiation therapy methods. One benefit of such combination methods is that use of an antibody molecule may permit a reduction in the chemotherapy and/or radiation dosage necessary to inhibit tumor growth and/or metastasis. As used herein, "radiotherapy" includes but is not limited to the use of radio-labeled compounds targeting tumor cells. Any reduction in chemotherapeu- tic or radiation dosage may benefit a patient by resulting in fewer and decreased side effects relative to standard chemotherapy and/or radiation therapy treatment (accordingly, quality of life may be higher where dosages of such compounds are reduced in cancer treatment). In this aspect, an antibody molecule (e.g., an anti-α3 G1 G2 antibody) may be administered or otherwise delivered prior to, at the time of, or shortly after a given round of treatment with chemotherapeutic and/or radiation therapy. Typically, an anti-α3 G1 G2 antibody is administered prior to or simultaneously with a given round of chemotherapy and/or radiation therapy in practicing such methods, which may be all rounds or less than all rounds. The exact timing of antibody administration typically will be determined by an attending physician based on a number of factors. In one exemplary aspect, an anti-α3 mAb can be administered or otherwise delivered about 24 hours before a given round of chemotherapy and/or radiation therapy and simultaneously with a given round of chemotherapy and/or radiation therapy.
Methods of the invention can be appropriate for application in conjunction with chemotherapy regiments using one or more anti-cancer cytotoxic agents/chemotherapeutic agents, including, but not limited to, cyclophosphamide, taxol (and other taxanes, such as docetaxel, paclitaxel, and the like), 5-fluorouracil, adriamycin, cisplatin, methotrexate, ox- aliplatin, irinotecan, topotecan, leucovorin, carmustine, streptozocin, cytosine arabinoside, mitomycin C, prednisone, vindesine, carbaplatinum, and vincristine. Additional chemotherapeutic and cytotoxic agents are described elsewhere herein and further suitable chemo- therapeutic and cytotoxic agents are known in the art. For a general discussion of cytotoxic agents used in chemotherapy, see, e.g., Sathe, M. et al., Cancer Chemotherapeutic Agents: Handbook of Clinical Data (1978) and the second edition thereof (Preston - 1982), and Cancer Chemotherapeutic Agents (Acs Professional Reference Book) (William Foye, Ed. 1995). Application of therapeutic methods of the invention also can be particularly suitable for those patients in need of repeated or high doses of chemotherapy and/or radiation therapy under current therapeutic regimens. Delivery of antibody molecules, alone or in combination with such agents, can provide a mechanism for reaching an at least as therapeutically effective outcome in a cancer patient with substantially lower amounts of chemotherapy and/or radiation therapy. In general, combination administration methods of the invention can comprise any suitable administration scheme, including coadministration (as separate compositions or a single composition wherein the ingredients are mixed or separated) or stepwise administration of the various active agents.
In another aspect, an antibody molecule is delivered in association with inhibitors, binding molecules, or antibodies against Ln-6 and/or antibodies that specifically target Ln5B, over Ln5A, or visa versa (or fragments thereof). Ln5B and related protein fragments are described in, e.g., Kariya et al., J Biol Chem. 2004 Jun 4;279(23):24774-84. Epub 2004 Mar 23). The invention further provides a method that also or alternatively comprises delivering antibodies against the β3 chain of Ln5 to a host, so as to reduce cell migration, invasiveness, etc. In a further aspect, inventive methods described herein can further include the delivery of an antibody against matrilysin, an antibody against CD44, or both.
Various other combinations with antibody molecules are described elsewhere herein (typically in the context of combination compositions) that can be similarly used in combination therapies of the invention and visa versa. In delivering antibody molecules, the amount or dosage range of the antibody molecule employed typically is one that effectively inhibits tumor growth, tumor cell invasiveness, and/or metastasis. An inhibiting amount of antibody that can be employed in such methods, for example, can range from generally between about 0.01 μg/kg body weight to about 15 mg/kg body weight, such as between about 0.05 μg/kg and about 10 mg/kg body weight, more specifically between about 1 μg /kg and about 10 mg/kg body weight, and even more particularly between about 10 μg /kg and about 5 mg/kg body weight.
Usually a daily dosage of active ingredient can be about 0.01 to 100 milligrams per kilogram of body weight. Ordinarily, about 1 to about 5 or about 1 to about 10 milligrams per kilogram per day given in divided doses of about 1 to about 6 times a day or in sustained re- lease form may be effective to obtain desired results. As a non-limiting example, treatment of Ln5-related pathologies in humans or animals can be provided as a daily dosage of antibody molecule(s), such as monoclonal, chimeric, and/or murine antibodies, in an amount of about 0.1 -100 mg/kg, such as 0.5, 0.9, 1 , 2, 3, 5, 7, 10, 12, 15, 20, 25, 30, 40, 50, 70, 90 or 100 mg/kg, per day, on at least one of day 1 , 2, 3, 4, 5, 6, 7, 10, 12, 14, 17, 21 , 24, 28, 30, 35, or 40, or alternatively, at least one of week 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20, or any combination thereof, using single or divided doses of every 24, 12, 8, 6, 4, and/or 2 hours. Other principles relevant to dosage are described elsewhere herein.
In general, antibody molecules, related compounds, and related compositions can be administered via any suitable route, such as an oral, mucosal, buccal, intranasal, inhal- able, intravenous, subcutaneous, intramuscular, parenteral, intertumor, intratumor, or topical route. Such peptides, related compounds, and compositions may also be administered continuously via a minipump or other suitable device. An anti-α3 antibody or other antibody molecule may be administered parenterally in dosage unit formulations containing conven- tional pharmaceutically acceptable carriers, adjuvants, and the like (e.g., stabilizers, disintegrating agents, anti-oxidants, etc. as described elsewhere herein). The term "parenteral" as used herein includes, subcutaneous, intravenous, intraarterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques and intraperitoneal delivery. Most commonly, a α3 G1 G2 antibody will be administered intravenously or subcu- taneously, in practicing therapeutic methods of the invention. Routes of injection also include injection into the muscle (intramuscular IM); injection under the skin (subcutaneous (s.c.)); injection into a vein (intravenous (IV)); injection into the abdominal cavity (intraperitoneal (IP)); and other delivery into/through the skin (intradermal delivery, usually by multiple injections, which may include biolistic injections). An anti-α3 antibody or other antibody molecule generally will be administered for as long as the disease is present, provided that the antibody causes the condition to stop worsening or to improve. An anti-α3 antibody or other antibody molecule typically is administered as part of a pharmaceutically acceptable composition as described elsewhere herein.
An anti-α3 antibody or other antibody molecule (or related surrogate/composition) may also be administered or otherwise delivered prophylactically in order to reduce the risk of developing cancer, delay the onset of the occurrence of an event in cancer progression, and/or reduce the risk of recurrence when a cancer is in remission. This type of method may be especially useful in patients wherein it is difficult to locate a tumor that is known to be present due to other biological factors. In one aspect, an antibody molecule (e.g., a cytotoxic antibody molecule conjugate) or related composition (e.g., a combination composition) is administered by regional perfusion therapy (wherein the agent or composition is delivered directly to target organs or areas affected by or at risk of being affected by cancer). For therapy, antibody molecules may be administered topically or parenterally, e.g. by injection at a particular site, for example, subcutaneously, intraperitoneal^, intravascu- larly, intranasally, transdermal^, or the like. Formulations for injection can comprise any suitable excipients, and typically will comprise (or be substantially composed of) a physiologically-acceptable medium, such as water, saline, PBS, aqueous ethanol, aqueous ethylene glycols, or the like. Water soluble preservatives which may be employed in such formulations include sodium bisulfite, sodium thiosulfate, ascorbate, benzalkonium chloride, chloro- butanol, thimerosal, phenylmercuric borate, parabens, benzyl alcohol, and phenylethanol. These agents may be present in individual amounts of from about 0.001 to about 5% by weight and preferably about 0.01 to about 2%. Suitable water soluble buffering agents that may be employed in these and other formulations described herein include alkali or alkaline earth carbonates, phosphates, bicarbonates, citrates, borates, acetates, succinates and the like, such as sodium phosphate, citrate, borate, acetate, bicarbonate and carbonate. Additives such as carboxymethylcellulose may be used as a carrier in amounts of from about 0.01 to about 5% by weight. A formulation can vary depending upon the purpose of the for- mulation, the intended treatment, and the like. A formulation may involve patches, capsules, liposomes, time-delayed coatings, pills, or may be formulated in pumps for continuous administration. Specific dosages can be determined empirically by standard methods. See, for example Harrison's, Principles of Internal Medicine, 1 1 th ed., Braunwald et al. ed, McGraw Hill Book Co., New York, 1987. In another aspect, two or more antibody molecules are delivered to a host, such as a human patient, to induce, promote, and/or enhance a beneficial physiological response, such as the reduction of cancer progression. As described elsewhere herein, in one aspect the invention provides compositions comprising a plurality (e.g., a "cocktail") of anti-G1/G2 antibody molecules. This and other combination compositions can be used in various inven- tive methods provided here and to prepare medicaments for the treatment of diseases, disorders, and conditions described here. Delivery of such combinations of antibody molecules is another feature of the invention.
In one aspect, inventive methods provided herein can comprise administering or otherwise delivering two different antibody molecules over a period of time, wherein the de- livery of such different antibody molecules overlap or do not overlap. For example, the in- vention provides a method of delivering two or more antibody molecules over a period of one month, the beginning of the therapy involving the second antibody molecule starting about 1 - 3 weeks (e.g., about 10 days) after the first delivery of the first antibody molecule or at any time when a significant immune response to the first antibody molecule develops in the host, such that the continued use of the first antibody molecule has become detrimental to the patient or ineffective. Such methods can be particularly advantageous when using humanized antibodies in the inventive therapeutic regimens described herein.
From the foregoing, it will be apparent that the invention provides various methods of treating cancer in a mammalian host, such as a human patient, comprising delivering (e.g., administering by administration alone or in a pharmaceutical composition of the invention or delivering via expression from a recombinant vector) an effective amount of an antibody molecule having any one or more of the above-described features (e.g., an antibody molecule derived from mAb 7B2) and that such methods may optionally be practiced in combination with additional anti-cancer therapies. In one aspect, such a method is performed to treat a human that is suffering from or at substantial risk of developing colon cancer. In another aspect, the practitioner performs the method to treat a human is suffering from or at substantial risk of developing a squamous cell carcinoma (for example, to treat a squamous cell carcinoma of the esophagus, cervix, bladder, oral cavity, or skin). In another aspect, the invention provides a method of treating non-small cell lung cancer in a human patient. In yet an- other aspect, the invention provides for the use of an antibody molecule, having any of the above described features (e.g., an antibody molecule derived from mAb 7B2, such as a humanized antibody or a fragment thereof or another antibody that competes with mAb 7B2, in the preparation of medicaments for the treatment of such diseases (e.g., for the treatment of colon cancer, non-small cell lung cancer, or squamous cell carcinoma). In another sense, the invention also provides a method for inhibiting the migration of cancer cells and/or other Ln5-associated endothelial cells by contacting such cells with an effective amount of an antibody molecule of the invention. The practice of such methods may be relevant to the treatment of other Ln5-associated disorders, diseases, and conditions (e.g., the treatment of polycystic kidney disease). The use of such antibody molecules in the preparation of medicaments for such diseases is another feature of the invention.
As indicated above, various methods and compositions effective in the treatment of cancer can be combined with the delivery of an effective amount of one or more antibody molecules to a subject in the treatment of cancer. Particular examples of such techniques and compositions are described in further detail here. For convenience, such methods and compositions are sometimes referred to as "combination methods," "combination compositions," and the like herein.
In one aspect, the invention provides a method of treating cancer that comprises coadministering an antibody molecule of the invention and one or more additional anti-cancer agents. The terms "coadministration," "coadminister," and the like herein refer to both simultaneous (or concurrent) and serial but related administration, unless otherwise indicated. Coadministration of agents can be accomplished in any suitable manner and in any suitable time. In other words, coadministration can refer to administration of an antibody molecule before, simultaneously with, or after, the administration of the secondary antineoplastic agent, at any time(s) that result in an enhancement in the anti-cancer response over the administration of solely the antineoplastic agent, antibody molecule, or both (i.e., over either independently). Words such as "co-deliver" and "co-delivered" are to be similarly interpreted, except in also encompassing delivery by other routes besides administration (e.g., expression of a protein from an administered nucleic acid, internal targeted production of an active anti-cancer agent from an administrated prodrug, etc.), unless otherwise stated or clearly contradicted by context.
In one exemplary aspect, the invention relates to combination compositions comprising an antibody molecule and one or more anti-cancer compounds, optionally in further combination with pharmaceutically acceptable carriers and agents (e.g., buffers, stabilizers, etc.). A large number of exemplary combinations have already been described herein. In general, any agent that has been described as being potentially useful in such a combination composition also may be used in a combination therapy protocol.
In another aspect, the invention relates to a combination method (combined therapy regimen) comprising delivery of an effective amount of an antibody molecule of the invention in combination with the application of one or more anti-cancer therapies.
In one aspect, the invention provides a combination method that comprises application of radiation or associated administration of radiopharmaceuticals to a patient in combination with one or more antibody molecules. In a related facet, the invention provides a composition comprising an effective combination of one or more antibody molecules and one or more radiopharmaceuticals.
The source of radiation in such methods can be either external or internal to the patient being treated (radiation treatment can, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that can be used in practicing such methods and included in such compositions include, e.g., radium, Cesium- 137, lridium-192, Americium-241 , Gold-198, Cobalt-57, Copper-67, Technetium-99, Iodide- 123, lodide-131 , and lndium-1 1 1 . Additionally useful radionuclides that can be incorporated in radiopharmaceuticals and used in such methods are discussed elsewhere herein (e.g., in the context of antibody molecule conjugates). In a particular aspect, such methods and compositions also further optionally include one or more radiation protectors. These drugs are designed to protect normal cells from radiation. One example is the intravenous drug amifostine (Ethyol). In another particular aspect, intensity-modulated radiation therapy (IMRT) is applied in combination with the delivery of one or more antibody molecules. IMRT delivers radiation therapy that is targeted to tumor shape, minimizing damage to healthy tissue. More particularly, IMRT allows radiation beams to be divided up and delivered in differ- ent intensities and directions to match the tumor's shape. Another similar technique, three- dimensional conformal radiation therapy, has been demonstrated to be effective in some situations and can be combined with antibody molecule therapeutic and prophylactic regimens. Pulsed delivery (gating) of radiation also or alternatively can be used in such methods, reducing the field of radiation by compensating for natural breathing patterns. In a further facet, antibody molecules are provided in association with the application of a radiogenic therapy. One example of such a therapy is the localized production of cytotoxic agents by radiation stimulation/activation of a prodrug or gene associated with a radia- tion-inducible promoter (such a gene may code for a cytotoxic protein, an enzyme that activates a co-delivered prodrug, etc.). Another example of such a radiotherapy agent is tar- geted auger-emitting radiolabeled molecules. These therapies can control cancer by delivering targeted radiation to specific receptor bearing cells. Auger electrons are emitted by radioactive isotopes (lodine-125 or lndium-1 1 1 ). The electrons have very short ranges and therefore have the potential to be delivered to specific sets of target cells, sparing healthy cells. In another exemplary method, a nucleic acid comprising a radiation-induced gene se- quence that codes for a protein that can be targeted by a cytotoxic agent is delivered in association with one or more antibody molecules. Radiation is applied to produce the protein and the cytotoxic agent delivered so as to provide a targeted therapy.
In further aspects, antibody molecules are delivered in connection with application of photodynamic therapy. In general, such therapies involve the delivery of a photosensitizing agent that makes cells more sensitive to light and, by doing so, causes cancer cells to be destroyed when a laser light is directed on a cancerous area. Thus, various prophylactic and therapeutic regimens of the invention also or alternatively can be combined with anti-cancer directed photodynamic therapy (e.g., anti-cancer laser therapy - which optionally can be practiced with the use of photosensitizing agent, see, e.g., Zhang et al., J Control Release. 2003 Dec 5;93(2):141 -50)). Rhodium compounds, for example, can damage DNA in living cells in a manner similar to platinum classic chemotherapy drugs, while remaining benign until irradiated with light.
Lasers also can be used in the performance of precise anti-cancer surgeries (e.g., where labeled antibody molecules have identified cancerous tissues and/or precancerous growths). Other forms of surgery also or alternatively can be applied in connection with the delivery of one or more antibody molecules. Anti-cancer surgical techniques (e.g., colectomy, proctocolectomy, polypectomy, prostatectomy, segmental resection, lobectomy, pneumonectomy, lumpectomy, mastectomy, etc.) are well known in the art and accordingly are not discussed in detail here (see, e.g., CANCER SURGERY, Harvey and Beatie (W. B. Saunders Company 1996); ADVANCED ONCOLOGIC SURGERY, Roh et al. Eds. (Mosby-Year Books, 1 st Ed. 1994); CANCER SURGERY, McKenna et al. Eds. (Lippincott Williams & Wilkins 1994); The M. D. ANDERSON SURGICAL ONCOLOGY HANDBOOK, Feig et al. (Lippincott Williams & Wilkins; 3rd Ed. 2002); and SURGICAL ONCOLOGY: CONTEMPORARY PRINCIPLES AND PRACTICE, Bland et al. (McGraw-Hill Professional; 1 st Ed. 2001 ). In a particular aspect, antibody molecule therapy and/or labeled antibody molecule diagnostic techniques is/are combined with anti-cancer cryosurgery. In another aspect, organs (such as ovaries or testicles) that make hormones may be removed in connection with antibody molecule anti-cancer therapy. In a further aspect, antibody molecule therapy is combined with the application of a bone marrow transplant and/or anti-cancer stem cell therapy. Stem cell transplantation (SCT), for example, may advantageously used in cancer treatment. The SCT may be autologous (the person's own cells that were saved earlier), allogeneic (cells donated by another person), or syngeneic (cells donated by an identical twin). SCT methods and related principles are known in the art (see, e.g., Georges et al., lnt J Hematol. 2003 Jan;77(1 ):3-14; Tabbara et al., Anticancer Res. 2003 Nov-Dec;23(6D):5055-67; Bhatia et al., Expert Opin Biol Ther. 2001 Jan;1 (1 ):3-15; Huugen et al., Neth J Med. 2002 May;60(4):162-9; Margolin et al., J Urol. 2003 Apr;169(4):1229-33; and US Patent 6,143,292). Bone marrow transplant is an even more well known method used in treatment of certain cancers (see, e.g., Thomas, Ann N Y Acad Sci. 1995 Dec 29;770:34-41 ; KoIb and Holler, Stem Cells. 1997;15 Suppl 1 :151 -8; Thomas, Semin Hematol. 1999 Oct;36(4 Suppl 7):95-103). Antibody molecules also can be delivered in association with application of other therapeutic methods such as anti-cancer sound-wave and shock-wave therapies (see, e.g., Kambe et al., Hum Cell. 1997 Mar;10(1 ):87-94); anti-cancer thermotherapy (see, e.g., US Patent 6,690,976), and/or anti-cancer neutraceutical therapy (see, e.g., Roudebush et al., Vet Clin North Am Small Anim Pract. 2004 Jan;34(1 ):249-69, viii and Rafi, Nutrition. 2004 Jan;20(1 ):78-82). Other methods include diet therapies (e.g., fasting therapy (which may be aided by anti-obesity agents or anti-appetite agents) or adoption of a high potassium, low sodium (saltless) diet, with no fats or oils, and high in fresh raw fruits and vegetables - see, e.g., A Cancer Therapy: Results of Fifty Cases, Max Gerson, Gerson Inst; 6th edition). Another technique that may be combined with antibody molecule anti-cancer therapy is insulin potentiation therapy, wherein low-dose insulin is given in conjunction with low-dose chemotherapy and antibody molecule anti-cancer therapy.
Antibody molecule anti-cancer methods also can be applied in conjunction with various adjunct therapies designed to ameliorate cancer-associated and cancer treatment- associated conditions, such as treatments for depression, treatments for pain (e.g., by deliv- ery of morphine or a morphine derivative), treatment for incontinence, treatment for impotence, etc.
Chemotherapeutic drugs may lack the ability to adequately penetrating tumors to kill them because these cells may be dead or lack a good blood supply. Anaerobic bacteria, such as Clostridium novyi, can consume the interior of oxygen-poor tumors. Such bacteria die when they come in contact with the tumor's oxygenated sides, meaning they are likely harmless to the rest of the body. The application of such bacteria and one or more antibody molecules represents another feature of the invention. Typically, such methods are practiced in further combination with a chemotherapeutic agent.
The inventive methods described herein also or alternatively can be practiced in connection with the delivery of one or more agents that promote access of an antibody molecule, related compound, or combination thereof to the interior of a tumor. Thus, for example, such methods can be performed in association with the delivery of a relaxin, which is capable of relaxing a tumor (see, e.g., US Patent 6,719,977). As another example of such a technique, a oc3 G1 G2 or related compound (e.g., an anti-idiotype anti-α3 G2 mAb or an immu- nogenic α3 peptide) can be bonded to a cell penetrating peptide (CPP). Cell penetrating peptides and related peptides (such as engineered cell penetrating antibodies) are described in, e.g., Zhao et al., J Immunol Methods. 2001 Aug 1 ;254(1 -2):137-45; Hong et al., Cancer Res. 2000 Dec 1 ;60(23):6551 -6; Lindgren et al., Biochem J. 2004 Jan 1 ;377(Pt 1 ):69-76; Buerger et al., J Cancer Res Clin Oncol. 2003 Dec;129(12):669-75; Pooga et al., FASEB J. 1998 Jan;12(1 ):67-77; and Tseng et al., MoI Pharmacol. 2002 Oct;62(4):864-72. Intratu- moral administration of α3 G1 G2s or vectors comprising antibody molecule-encoding or related molecule-encoding nucleic acid sequences also or alternatively can be used to facilitate therapeutic regimen aspects of the invention.
Additional target Ln5-binding molecules and molecules that are involved with Ln5- influenced aspects of cancer progression and, accordingly, are advantageous targets for secondary molecules in the context of combination compositions and/or combination delivery methods (or as being one of the targets in a bispecific anti-α3 G1 G2 antibody as described elsewhere herein) include α6β1 integrin, α3β1 integrin, α2β1 integrin, α6β1 integrin, laminin- 6, laminin-7, EGF-R, type VII collagen, fibulin-1 , fibulin-2, Rho GTPases, BP180, syndecan- 4, nidogen-1 , phosphorylated hsp-27, p300, a cytokeratin, and other matrix metallopro- teinases (e.g., MMP-1 , MMP-2, MMP-9, and MMP-14 (which also known as Membrane-type matrix metalloproteinase 1 (MT1 )), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1 ) and TIMP-2, E-cadherin, bone morphogenic protein-1 (BMP-1 ), and the 67 kDa laminin receptor. Thus, in one aspect, the invention provides a method of reducing a cancer progres- sion aspect (e.g., cancer cell migration) in a human patient in need thereof comprising delivering an antibody molecule and an antibody specific for one or more of these non-similar molecules to the patient in amounts and under conditions such that cancer progression is detectably reduced in the patient. Additional types of such molecules are discussed elsewhere herein and/or are known in the art. The invention also provides methods for the identification of, and diagnosis of invasive cells and tissues, and other cells targeted by antibody molecules, and for the monitoring of the progress of therapeutic treatments, status after treatment, risk of developing cancer, cancer progression, and the like.
In one example of such a diagnostic assay, the invention provides a method of di- agnosing the level of invasive cells in a tissue comprising forming an immunocomplex between an antibody molecule and potential Ln5 containing tissues or components thereof, and detecting formation of the immunocomplex, wherein the formation of the immunocomplex correlates with the presence of invasive cells in the tissue. Such "contacting" can be performed in vivo, using labeled isolated antibodies and standard imaging techniques, or can be performed in vitro on tissue samples.
Antibody molecules can be used to detect α3-containing peptides and peptide fragments in any suitable biological sample (e.g., a tissue sample from a human patient, a cell culture, etc.) or other composition by any suitable technique. Examples of conventional immunoassays provided by the invention include, without limitation, an ELISA, an RIA, FACS assays, plasmon resonance assays, chromatographic assays, tissue immunohistochemistry, Western blot, and/or immunoprecipitation using an antibody molecule. Thus, in one aspect, the invention provides a method of detecting/assaying α3-containing peptides in a composition comprising adding to the composition one or more antibody molecules and thereafter adding labeled or detectable secondary antibodies (e.g., anti-human antibody antibodies or anti-α3 Ab antibodies) to the composition so as to detect whether any proteins or structures in the composition are bound by the secondary antibody. In another aspect, an antibody molecule conjugated with a detection-facilitating agent (a fluor, an enzyme, a radionuclide, etc.) may be used for such diagnostic assays. Anti-α3 antibodies of the invention may be used to detect α3 and α3-containing peptides in biological samples obtained from humans or in human tissues in vivo. Suitable labels for the antibody and/or secondary antibodies used in such techniques have been disclosed supra, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include strepta- vidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbellifer- one, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive materials include 1251, 131 1, 35S, and 3H.
Ln5 oc3 peptides also can be assayed in a biological sample by a competition immu- noassay utilizing α3 peptide standard(s) labeled with a detectable substance and an unlabeled anti-α3 G1 G2 antibody, for example. In such an assay, the biological sample, a labeled oc3 peptide standard(s) and an anti-α3 G1 G2 antibody are combined and the amount of labeled α3 standard bound to the unlabeled antibody is determined. The amount of α3 peptide in the biological sample typically is inversely proportional to the amount of labeled α3 standard bound to the anti-α3 G1 G2 antibody.
The antibody molecules are particularly useful in the in vivo imaging of tumors, as briefly mentioned elsewhere herein. In vivo imaging of tumors associated with α3 can be performed by any suitable technique. For example, 99Tc or another gamma-ray emitting isotope can be used to label α3 G1 G2 antibodies in tumors or secondary labeled (e.g., FITC labeled) antibody: α3 G1 G2 complexes associated with tumors and the labeled antibodies imaged with a gamma scintillation camera (e.g., an Elscint Apex 409ECT device), typically using a low-energy, high resolution collimator or a low-energy all-purpose collimator. Stained tissues can then be assessed for radioactivity counting as an indicator of the amount of Ln5 α3-associated peptides in the tumor. The images obtained by the use of such techniques can be used to assess biodistribution of α3-associated peptides in a patient, mammal, or tissue, for example in the context of using α3 or α3-containing peptides as a biomarker for differentiation of epithelial cells (e.g., in the context of cancer progression toward malignant growth) or the presence of invasive cancer cells. Variations on such techniques can include the use of magnetic resonance imaging (MRI) to improve imaging over such gamma scintilla- tion camera techniques. Similar immunoscintigraphy methods and principles useful in performing such techniques are described in, e.g., Srivastava (ed.), Radiolabeled Monoclonal Antibodies For Imaging And Therapy (Plenum Press 1988), Chase, "Medical Applications of Radioisotopes," in Remington's Pharmaceutical Sciences, 18th Edition, Gennaro et al. (eds.), pp. 624-652 (Mack Publishing Co., 1990), and Brown, "Clinical Use of Monoclonal Antibodies," in Biotechnology and Pharmacy 227-49, Pezzuto et al. (eds.) (Chapman & Hall 1993) (incorporated in its entirety). Such images also can be used for targeted delivery of other anti-cancer agents, examples of which are described herein (e.g., apoptotic agents, toxins, or CHOP chemotherapy compositions). Moreover, such images also or alternatively serve as the basis for targeting surgical techniques to remove tumors. Furthermore, such in vivo imaging techniques may allow for the identification and localization of a tumor in a situation where a patient is identified as having a tumor (due to the presence of other biomarkers, metastases, etc.), but the tumor cannot be identified by traditional analytical techniques. All of these methods are also features of the invention. Diagnostic kits can also be supplied for use with an antibody molecule, such as a conjugated/labeled anti-α3 mAb, for the detection of a cellular activity (e.g., epithelial cell migration, carcinoma invasiveness, etc.) or for detecting the presence of α3 peptides in a tissue sample or host. In such diagnostic kits, as well as in kits for therapeutic uses described elsewhere herein, an antibody molecule typically is provided in a lyophilized form in a con- tainer, either alone or in conjunction with additional antibodies specific for a target cell or peptide. Typically, a pharmaceutical acceptable carrier (e.g., an inert diluent) and/or components thereof, such as a Tris, phosphate, or carbonate buffer, stabilizers, preservatives, bio- cides, biocides, inert proteins, e.g., serum albumin, or the like, also are included (typically in a separate container for mixing) and additional reagents (also typically in separate con- tainer(s)). In certain kits, a secondary antibody capable of binding to the anti-α3 G1 G2 antibody or other antibody molecule, which typically is present in a separate container, also is included. The second antibody is typically conjugated to a label and formulated in manner similar to the anti-α3 G1 G2 antibody or other antibody molecule.
In another aspect of the invention, a method for identifying a compound useful for cancer treatment comprising screening candidate compounds for binding to hl_n5α3 in any part of residues 1064-1077 thereof and assessing such an identified compound in one or more anti-cancer activity assays. Such a method may also or alternatively comprise determining if the compound binds α3 within any part of (a) residues 989-1008, (b) residues 1082- 1090, or (c) both (a) and (b). In another aspect, the invention provides a method of identify- ing a compound useful for cancer treatment comprising also or alternatively screening candi- date compounds for the ability to compete with mAb 7B2 or a 7B2-related antibody molecule to bind Ln5 or a portion thereof and assessing such an identified compound in one or more anti-cancer activity assays. Such methods may be used also in preparing a pharmaceutical composition. For example, in one aspect the invention provides a method of preparing a pharmaceutical composition that comprises testing whether an active ingredient thereof binds to hl_n5 in any part of residues 1064-1077 thereof. Such methods may be used for quality control standards (e.g., "release assays") and the like. In another aspect, the invention provides a method for generating pharmacologically active molecules that bind to such regions of G2 and/or compete with mAb 7B2 for binding to G2 (or Ln5). Such molecules may be, e.g., G2-binding peptides, small molecules, or antibody-like molecules. Exemplary antibody-like molecules that may be so produced according to this aspect include G1/G2-specific affibodies, trinectins, monobodies, anticalins, and antibody mimetics. Such molecules are known in the art and described generally in WO2005040219 and references cited therein.
EXEMPLARY METHODS AND DATA The following exemplary experimental methods and data are presented to better illustrate various aspects of the invention, and related illustrative enabling technology, but in no event should be viewed as limiting the scope of the invention.
Example 1 - Analysis of 7B2 and BMI 65 Binding to Ln5 and Fragments Thereof To compare the binding of mAbs 7B2 and BM165 to Ln5, we conducted the following ELISA binding study.
Nunc-ELISA plates were coated with 1 μg of rl_N5, an Ln5 gamma2 domain 3 (γ2D3) peptide, α3G1 G2, α3G1 G2G3, α3G4G5, or a 100 kDa fragment of Ln5β3) over night at 4C. Plates were washed and blocked in blocking buffer for 15min and washed again. 100μl su- pernatant of mAb 7B2 and purified mAb BM165 (1 μg/ml) were applied and incubated for 1 h. After five washes, 1 :2000 diluted HRPO-labeled gt-anti-mouse was added and incubated for 45min. After five washes, bound antibody was detected by adding TMB-substrate. The color reaction was stopped with 3M H3PO4 and the OD was measured at 450nm to determine if binding occurred. The results of this experiment are shown in Table 2 (when binding was seen it is given a "+" and when no binding was seen it is indicated with a "-").
The results of these experiments demonstrate that both 7B2 and BM165 bind to a region of α3, which in the smallest version can be described as G1 G2. However, additional experiments provided herein demonstrate that 7B2 and BM165 bind to unique antigenic determinant regions within G1 G2.
Example 2 - Cross-Competition Ln5α3 Binding Studies with 7B2 and BM165 To determine whether 7B2 and BM165 cross-compete for binding to recombinant human laminin-5 (rl_N5)
Biotinylated versions of mAbs 7B2 and BM165 were prepared using standard techniques. Nunc-ELISA plates were coated with 0.25μg/well of rl_N5 in 0.1 M carbonate/bicarbonate buffer, pH 9, over night at 4<€. Plates were washed three times in PBST (10 mM Potassium phosphate, 150 mM NaCI, pH 7.5, 0.05 % Tween-20) and blocked 90 minutes at room temperature ("RT") in BSA-PBS (1% bovine serum albumin in PBS buffer). The plates were washed again with PBST before antibodies are added.
The putative competitive ("competitive") biotinylated antibodies were mixed together as follows. Competitive Ab 5μg/well, biotinylated Ab 0.05 μg/well, diluted in BSA-PBS and incubated for 30 minutes at room temperature.
After three washes with PBST, binding detection was carried out using the AB system (biotin/avidin system, Elite Standard Kit, cat. PK-1600, Vector Laboratories Inc, Burlin- game, CA) (incubation time of 30 minutes at RT). Subsequently, the media was subjected to three washes with PBST before development using ABTS-peroxidase substrate concentrate (1 Ox = 1 mg/ml): ABTS diluted in 0.1 M Na-citrate, pH 5 (diluted for assay just before use, 1 ml-> 10 ml with Na-citrate buffer and add 2 μl 30 % hydrogen peroxidase (ABTS = 2,2'- azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)). Optical density values were measured in an ELISA reader (Absorbance A405) after 30 minutes of color reaction and the value obtained with biotinylated mAb alone (without competing mAb) was set to 100%. The competi- tion was then measured by expressing percentage binding of the biotinylated mAb in presence of the competing mAb.
The results of this experiment are shown in Tables 3 and 4: Table 3 - % of Biotinylated BM165 Binding to rLnδ
The results of these cross-competition experiments demonstrate that mAbs 7B2 and BM165 bind to different epitopes in hl_n5.
Example 3 - Cross-competition Analysis of mAbs 7B2, CM6, RG13, and BM165 The above-described antibodies also were used to perform additional cross- competition studies using standard methods (as briefly described above) involving biotinylated BM165 or 7B2. Plates were coated with rl_N332 (0.25 g/well). The results of these experiments are shown in Tables 5 and 6:
Table 5 - % binding of biotinylated BM165 to rLn332
The results of these cross-competition experiments demonstrate that mAb 7B2 binds an epitope not recognized by BM165, CM6, and RG13. Some competition for RG13 in BM165 binding can be seen indicating at least some overlap in binding site between these two mAbs. The CM6 mAb does not interfere with binding of 7B2 or BM165, this can be due to difference in epitopes or that CM6 may not be active in the assay.
Example 4 - Cell Surface Binding Analysis of 7B2 Human SCC-25 squamous carcinoma cells (ATCC) produce large amounts of laminin-5. SCC-25 cells were therefore used to determine if 7B2 binds at the cell surface of tumor cells.
SCC-25 cells were grown to subconfluency in DMEM supplemented with 10 % FCS, and scraped off from the surface the tissue culture flask using a rubber policeman. The re- leased cells were collected with ice-cold PBS and pelleted by centrifugation. After washing in PBS, the cells were resuspended in PBS supplemented with 2 % FCS filtered to obtain a single cell suspension through a BD Falcon Cell Stainer (Ref 352340), and counted before dividing into plates (Plate U-bottom 353077 Falcon) with 1 x105 cells/well for staining with the below-indicated antibodies. Each antibody was added at 10μg/ml and incubated 30 minutes at 370C. Cells were pelleted by centrifugation and washed twice with PBS-B (PBS with 0.5 % w/v BSA) before secondary Ab was added as 1 :50 dilution in PBS-B. The detection Ab's used were Anti-Mouse APC (715-136-150 (Jackson ImmunoResearch F(ab)2 Anti -Mouse IgG (H+L)) (incubation was performed for 30 min. at 370C (light protected)). Cells were thereafter washed three times with PBS-B and subjected to flow cytometry analysis using a BD FACS. Secondary antibody was also used for background control in order to eliminate unspecific cell binding and anti-γ2D3 mAb 5D5 was used as a positive control.
Likewise, in SW480 human colon cancer cells (also available through ATCC), cells grown to subconfluency, scraped off with rubber policeman, and stained with mAb followed by detection by flourocrome-labelled secondary antibody and analysis by flow cytometry. The results of these experiments are shown in Fig. 1 . The data set forth in Fig. 1 demonstrate that only 7B2, as well as the positive control mAb (anti-γ2D3 mAb 5D5), were able to bind to the cell surface. BM165 cannot be seen to bind to the cell surface, indicating the epitope is not accessible for BM165.
Example 5 - Study of pAb and 7B2 Effect on Cell Adhesion by ECIS Analysis
Measurement of adhesion using electrical impedance (Electric Cell-substrate Impedance Sensing ("ECIS")) was preformed as follows. For cell electrical sensing, we used the RT-CES equipment from ACEA Biosciences. The basic unit in the RT-CES system is an array with 16 wells, in which each well has the size of a well in a 96-well plate). In the bottom of each well are two interdigitating gold electrodes connected to an electrical pulse generator that delivers 1 OmV at different frequencies. The resulting currents are measured and the impedance derived. A parameter Cl (cell index) is calculated according to the formula: Cl = max (RCeiι(fι)/Ro(fι) - 1 )- Rceiι(fι) and R0(f,) are the frequency-dependent electrode resistance without or with cells present in the well, respectively at three different frequencies (1 OkHz, 25kHz, 5OkHz). CI=O, when no cells are present (ie RCΘn(fι) = Ro(f.)- Overall it can be stated that Cl is a measure of the number of attached cells or/and their electrode contact area.
Wells were coated with laminin 5 (LN5) 5μg/ml in water for 1 h at 37^ followed by 2x wash using water and blocked for 30 min with 0,5% BSA in PBS. Finally the wells are washed twice with D-MEM and 50 up medium (D-MEM with 10% FBS) was added to the wells together with antibody (50 μg/ml as max dose or graded doses for IC50 determination) and the background Cl measured. The experiment was started by adding 100 μl_ cell suspension with 25.000 cells and follow Cl. The polyclonal antibodies (pAbs) to different regions of Ln332 was raised in rabbits immunized with recombinantly produced fragments of Ln332. Two rabbits were immunized in parallel and the specificity for Ln332 was verified in a standard ELISA (essentially performed as in example 1 ). In addition, the unspecific binding was characterized by immunohistochemistry (IHC) with the protocol stated in example 7. Each rabbit gave rise to a pAb where the two raised using the same immunogen always performed equally in the assays.
The results of these experiments are shown in Table 7
Table 7 - the Influence on Cell Adhesion ofpAbs Against Different Regions of Lnδ
These results indicate that the inhibition of cell adhesion achieved by polyclonal antibody to oc3G1 G2 is equipotent to that achieved by polyclonal Abs to the full molecule. This suggests that the most important part of the molecule for mediating adhesion of these cancer cells is present in the G1 G2 part of Ln5. This result may be somewhat unexpected given previous reports in the literature (particularly in previously published patent documents). The IC50 values for BM165 and 7B2 inhibition of cell adhesion to a LN5 coated surface for three different tumor cell lines also were determined using standard methods. The results of these experiments are set forth in Table 8.
The testing of the two mAbs 7B2 and BM165 shows that in the two cell lines (A431 and MDA-MB231 ) 7B2 is more potent than BM165 in blocking adhesion. 7B2 and BM165 showed more similar ability to inhibit adhesion in SCC-25 cells (though 7B2 still showed a slightly greater inhibition in these cells as well).
Example 6 - Study of 7B2 Effect on Tumor Cell Migration
The effect of 7B2 on cell migration was studied using a standard transwell assay. Briefly, a 24-well FluoroBlock insert system (BD Falcon) with pore size of 8 μm was used. The membrane was coated overnight with 0.5 μg/ml laminin-5 in PBS at +4 0C - total of 0.2 μg protein per well. MDA-MB231 tumor cells were grown till 80% confluence and detached with 10 mM EDTA (Versene solution, Invitrogen/Gibco BRL), spun at 400 x g for 7 min., and re-suspended in DMEM with 10 mM HEPES. The cells were then incubated with 50 μg/ml of the indicated antibodies for 30 min at room temperature prior to loading into the top com- partment of a transwell. 2x105 cells per well were used in the assay. The lower compartment of the transwell was filled with DMEM containing 50 μg/ml of the indicated antibodies. All treatments were done in triplicate; several independent experiments have been performed. The cells were allowed to migrate for 16-18 hours at 37 0C in a humidified incubator in the presence of 5% CO2. Cells migrated through the membrane into the lower compart- ment were stained directly in the transwell chamber with Calcein AM (Molecular Probes) for 30 min in the CO2 incubator. The staining solution was replaced with DMEM and the plates were scanned using a Fluoroskan Ascent (Thermolabs).
Migration of the MDA-MB231 cells through a LN5 coated membrane without inhibitors was set as 100%. In the absence of LN5 (uncoated membrane) migration did not exceed 10% (basal migration). The 7B2 mAb completely inhibited LN5 dependent migration (to below the basal migration level). Polyclonal antibody against G1 -G2 domains of the LN alpha3 chain inhibited migration to the same extend as 7B2. Meanwhile, P3E4 and P3H9-2 (dis- cussed above), showed only minor inhibitory effect (70-80% remaining migratory activity). Data is presented as mean ± SD.
At 50 ug/ml no difference could be seen in efficacy between mAb's 7B2 and BM165, nor with the rabbit pAb's generated to G1 G2, or pAb's generated to the full LN5 molecule, all of which completely block the migration of the cancer cells.
This Example demonstrates mAb 7B2 is capable of blocking tumor migration as effectively as polyclonal antibodies to G1 G2 and complete Ln5.
Example 7 - Effect of 7B2 on Human Tumor Cell Growth in a Mammalian Model To test whether 7B2 is effective at blocking tumor growth, in vivo, a mouse model system with human cancer cells was used.
Female NMRI nu-nu mice were obtained from Taconic Europe (Denmark). The animals were approximately 8-10 weeks old on the arrival and were allowed to acclimatize for at least one week before start of experiments. The mice were housed in a standard ani- mal facility where the light was controlled on a 12 hr light-dark cycle, and the animals were given free access to food and drinking water. The animals were observed daily for clinical signs and their body weights were recorded regularly.
A431 , a human epidermoid carcinoma cell line, was obtained from American Type Culture Collection (ATCC #CRL-1555). The cell line was cultured in Dulbecco's modified Ea- gle's medium (DMEM) with GlutaMAX™ I supplemented with 4500 mg/L D-Glucose, 10 % heat-inactivated fetal calf serum (FCS), and 5 % Penicillin-Streptomycin. The cell line was grown in 175 cm2 (T175) Nunc Easy Flasks at 37 5C with 5 % CO2. When confluent, cells were passaged using 0.25 % Trypsin with 1 mM EDTA. Before injection, cells were harvested by trypsin collected in full medium and centrifuge, resuspended in PBS and filtered to ensure single cell suspension though BD Falcon Cell Stainer ( Ref 352340) filters. A single- cell suspension of 2x105 viable tumor cells in 0.2 ml PBS was injected subcutaneously into the right flank of the animals at day 0. Subsequently, tumor sizes were measured as two perpendicular diameters two times a week throughout the experiments, and the tumor vol- π ume was calculated by using the following formula: — dx ■ d2 ,where dλ < d2 , d represents
6 the two perpendicular diameters. For early treatment, selected animals (10 per group) were thereafter treated with 7B2, BM165, or PBS (as control) from day 3.
For treatment of established tumors, tumors were grown to an average size of 77 mm3 (day 14) before mice were randomized in three groups of 10 mice for treatment with 7B2, Erbitux (hEGFR antibody) or PBS (as control). First antibody dosage was 50 mg/kg, injected Lp., in 0.05 ml/10 g PBS. Dosage during treatment was 25 mg/kg, administered by Lp. injection, in 0.05 ml/10 g PBS.
For each group of animals a curve of the tumor volumes during the experiment was plotted against time. The termination criterion was a tumor volume of 1000 mm3 or more than 20 % weight loss from time of cell inoculation. The data from the last day of the experiment was subjected to statistical evaluation using Mann-Whitney U-test to evaluated the significance of the treatments on tumor volume compared to control. Significance is defined as p<0.05.
The results of these experiments are shown in Fig. 2 (early treatment of A431 xeno- graft tumors with 7B2 and BM165), Fig. 3 (same, but in a second experiment), and Fig. 4 (treatment of established tumors with 7B2 or Erbitux). The results shown in Fig. 2, demonstrate that both 7B2 and BM165 give a clear reduction in tumor volume at day 38, with BM165 giving p = 0.029 and 7B2 giving p = 0.023. At this dosing no difference can be seen in efficacy between the two mAbs 7B2 and BM165. However, in the study shown in Fig. 3, the significant inhibition of tumor volume is only evident for 7B2 at day 38 with a p=0.028, whereas BM165 having a trend for inhibiting tumor volume only get p=0.1 13 at day 38. In the results shown in Fig. 4, both Erbitux and 7B2 give a marked reduction of tumor volume at, e.g., day 35. Where 7B2 has p = 0.005 and erbitux has < 0.001 .
Example 8 - lmmunohistochemistry Staining of Different Tissues With 7B2 and Comparison to Other mAbs and pAbs
To identify tissues where 7B2 and other Abs bound, standard immunohistochemistry studies were performed. Briefly, paraffin sections (4 μm) were deparaffinized in xylene and re-hydrated. Heat-induced antigen retrieval was performed in a microwave oven using
Tris/EDTA buffer, pH 9. Endogenous peroxidase and biotin was blocked by treatment with 0.5 % hydrogenperoxide and Biotin Blocking System (DAKOcytomation), respectively. Sections were preincubated in 4 % casein (Tryptone Casein Peptone, Amresco) in TBS for 60 min. to block non-specific binding, and then incubated overnight at 40C in the same solution containing 1 μg/ml antibody as indicated in the table. Antibody binding was detected with biotinylated anti-rabbit IgG (Jackson) for pAbs and with anti-mouse IgG (Jackson) for mAbs and Tyramid Signal Amplification system NEL700 (NEN). Sections were developed with dia- minobenzidine (DAB), counterstained with Hematoxylin, dehydrated in xylene and mounted in Pertex. The results of these experiments are shown in Table 9.
Example 9 - Determination of Antigenic Determinant Region for 7B2 by HxMS
Amide hydrogen-deuterium exchange is a technique that, among other, is suitable for epitope mapping. The method involves allowing both the immunogen and the antibody to become deuterated by separately incubating them in D2O and thereafter combining them in order to form the antibody:immunogen complex. The complex is transferred back to normal H2O, thus exchanging the deuterium back to normal hydrogen and only the deuterium trapped at the contact interface will remain. The deuterium exchange reaction can be quenched and the deuterium label detected by mass spectrometry since deuterium will shift the mass to higher values. Furthermore, the deuterium label can be sub-localized to specific regions in the protein by pepsin digestion of the protein under quench conditions and subsequent mass spectrometric detection of the resulting peptides (The HXMS technique has been reviewed recently in Wales et al., (2006) Mass Spectrom. Rev. 25, 158-170). Deuterated peptides showing a mass increase are part of the contact area for the antibody. By HxMS, the peptides retaining deuterium after having bound 7B2 on alpha 3 G2 were found to represent an antigenic determinant region (binding region) for 7B2.
To perform the HxMS technique, the 7B2 antibody was coupled covalently to protein G sepharose beads (Sigma) in order to ease handling and for removing the antibody prior to mass spectrometric analysis of the immunogen. The coupling reactions were performed essentially as in Baerga-Ortiz et al.,(2002) Protein science 1 1 , 1300-1308. All exchange reactions were performed in 50 mM Na-phosphate pH 6.5, 50 mM NaCI in either H2O or in D2O (94% final deuterium in the buffer). The quench solution consisted of 22% (v/v) 1 .35 M tris(2-carboxyethyl)phosphine hydrochloride (adjusted to pH 2.5 using NaOH) and 78% (v/v) 0.5% formic acid. For each reaction, 400 pmol immunogen and 1200 pmol antibody coupled to protein
G beads was used (ie a 6x surplus of antibody binding sites). The immunogen and antibody were each diluted 20 times into the deuterated buffer and allowed to in-exchange deuterium for 600 sec. The deuterated immunogen and antibody was then combined and incubated for 60 sec allowing the complex to form. The buffer was removed from the beads and the back- exchange to normal hydrogen was initiated by placing the beads in H2O buffer and then incubating for either 600 sec or 1800 sec. The back-exchange was terminated by spinning off the buffer from the beads and adding the quench solution, which will, apart from quenching the exchange reaction, also facilitate the elution of immunogen from the antibody. The mixture was incubated for 2 min on ice before spinning off the quenched reaction. The samples were snap-frozen in liquid nitrogen and stored at -800C until sample analysis.
The samples were run on a high pressure liquid chromatography-mass spectrometry system where the entire plumbing system has been immersed in an ice bath as recently described in detail in Rand et al., (2006) J. Biol. Chem. M602968200. Briefly, the samples were injected directly onto a cooled pepsin column and allowed to digest for 3 min before flushing them onto a micro-column (approximately 2-μl bed volume of Poros 20R2) for desalting for 3 min at 250 μl/min in 0.05% trifluoroacetic acid. Chromatographic separation of peptides was achieved on an analytical C18 column (Luna, Phenomenex) and peptides were eluted into an electrospray ion source at 50 μl/min by a 10-min gradient of 10-40% acetonitrile in 0.05% trifluoroacetic acid. Positive ion-electrospray ionization mass spectra of eluted peptides were acquired on a LCT mass spectrometer (Waters Inc.).
Peptides of α3G1 G2 were identified in separate experiments using standard MS/MS methods. Average masses of peptide isotopic envelopes were determined from continuum data using the MagTran software (see Zhang et al., Journal of the American Society for Mass Spectrometry 9, 225-233). A total of 38 overlapping peptides were used for the analysis of α3G1 G2. Major gaps in the peptide map occur at the N-glycosylation sites. Analysis showed that most peptides representing the major part of α3G1 G2, as expected, do not retain deuterium upon 7B2 binding. Only a few specific regions did retain deuterium upon 7B2 binding and these regions thus represent the binding site on α3G1 G2 for the 7B2 antibody. Peptides comprising α3 amino acids 1064-1077, 1082-1090, 989-1008 and 989- 1016 specifically retained deuterium upon 7B2 binding (989-1016 and 1009-1016 were weakly deuterated, suggesting that these represent only a part of the antigenic determinant region bound by 7B2). However, since the peptide 1009-1016 did not retain deuterium, the deuterium label in peptide 989-1016 can be localized to residues 989-1008. The antigenic determinant region for 7B2 on α3G1 G2 was thus more specifically determined to be located within residues 989-1008, 1064-1077 and 1082-1090. Although not all residues involved in forming the antigenic determinant region are close in the primary structure, they are situated close in tertiary structure as analyzed using a modeled structure of α3G2 (not shown).
As noted above, of these regions, 1064-1077 was deuterated the strongest, suggesting that the main part of the 7B2 epitope is located in this region and that the other re- gions are more peripheral in the epitope.
This experiment demonstrates that mAb 7B2 binds to a unique region in α3G2. Other antibodies, antibody molecules, and other relevant molecules (e.g., small molecule pharmacophores) that bind to all or part of this region of G2 can be generated using standard techniques and screened for biological effects.
Example 10 - Testing of 7B2 and BM 165 murine mAbs for the antitumor effect on human squamous cell carcinomas
Anti-LN-5 antibodies (7B2 and BM165) can be evaluated as anti-cancer agents using an animal model with mice having a xenograft tumor established before treatment initia- tion. In the model, normal human keratinocytes are transformed using over expression of Ras and IKB, and the transformed keratinocytes are placed on a dermal equivalent, and transplanted to mice. The model resembles histologically an aggressive human SCC of skin. Thus, tumors are invasive into the underlying dermis and muscle. In this model, the tumor environment can be more natural, and the tumor more integrated in the tissue than traditional s. c. tumors. See also Marinkovich, Nature Review Cancer 2007;7:370-380.
The study is carried out by producing 30 dermal equivalents containing human squamous carcinoma cells created using Ras/IKB overexpression as described in Dajee et al. (Nature 2003; 421 , 639-43). Tumor growth of carcinoma xenografts is analyzed after transfer to immunodeficient mice in the presence of systemic laminin inhibiting antibodies. Keratinocyte isolation
Keratinocytes are isolated from skin sections using serum-free keratinocyte culture medium. They are expanded one passage, prior to exposure of high titer retroviral super- natants. Both Ras and IKBa expressing retroviral supernatant are used to treat cell cultures. Verification of Ras/IKB overexpression is determined through Western blot of total cell lys- ates made from cell cultures. Following successful verification of Ras/IKB overexpression, keratinocytes are cultured for a brief 3-day recovery period, before seeded atop human devitalized dermis.
Skin equivalent production
Dermis sections are obtained from autopsies collected by the National Disease Re- search Interchange, Philadelphia, PA, USA with consent. Dermis undergoes a stepwise treatment prior to grafting, including freeze-thaw cycles in glycerol in pregnated gauze, a one month treatement with PBS to induce epidermal separation, and incubation in antibiotic solution for extended periods of time. Finally the dermal sections are tested for sterility prior to use in graft cultures. Once dermal sections production is complete and keratinocytes are seeded atop the sections, the cultures are maintained for two weeks in DMEM with 10% fetal bovine serum and multiple growth factors. At the later phases of the culture, the skin sections are lifted to the air-fluid interphase for epidermal stratification. After two weeks, the dermal equivalents are grafted to mice. Each equivalent is 1 .5 by 1.5 cm. Animal experiments SCID mice are utilized as an in vivo models to study the effects of BMZ proteins on human skin development and tumor formation.
Graft recipient mice are anesthetized with Avertin, and, after the anesthesia has taken effect, the mice are shaved. Using sterile technique, a 1.5 by 1 .5 cm section of mouse skin is removed down to fascia from the dorsum of each mouse. Skin equivalent containing SCC cells are layered onto the graft bed, the wound is dressed with Bactroban ointment mixed with 2% lidocaine cream (to promote post anesthetic anesthesia), Adaptic, Telfa pad and Coban wrap The APLAC regulation in which animals cannot be kept outside the animal facilities> 12 hours is observed. Post-anesthetic recovery requires approximately two hours. Surgery sites require approximately two weeks for healing. Grafted mice are monitored daily after initial observation, during post-anesthetic recovery. Animals are monitored daily. After this time, the mice are monitored twice weekly until the wound is completely healed. The site of transplantation is chosen to be the midline dorsal area of the mouse to prevent the recovering animal from scratching its wound. Additionally, after surgery, each mouse is housed separately to prevent manipulation of the wound by other mice. Antibody treatment and tumor assessment
The scid/scid mice is injected with with antibody, 1 mg each mouse, per week for four weeks. Each week, 0.2 cc of antibody at a concentration of 5 mg/ml in sterile PBS is injected intraperitoneal^ to each mouse. 10 mice receive antibody BM165 (positive control), 10 mice receive antibody 7B2, 10 mice receive sterile PBS with no antibody. Tumors are meas- ured twice weekly, and at the end of the time period, mice are euthanized and tumors are harvested, weighed, and then examined microscopically.
All references, including publications, sequence database records, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law), regardless of any separately provided incorporation of particular documents made elsewhere herein.
The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate).
The description herein of any aspect or embodiment of the invention using terms such as "comprising", "having," "including," or "containing" with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of", "consists essentially of", or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).
All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.
The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents. This invention includes all modifications and equivalents of the subject matter recited in the claims and/or aspects appended hereto as permitted by applicable law.
Priority Applications (2)
|Application Number||Priority Date||Filing Date||Title|
|Publication Number||Publication Date|
|WO2008005828A2 true WO2008005828A2 (en)||2008-01-10|
|WO2008005828A3 WO2008005828A3 (en)||2008-06-26|
Family Applications (1)
|Application Number||Title||Priority Date||Filing Date|
|PCT/US2007/072457 WO2008005828A2 (en)||2006-06-30||2007-06-29||PHARMACEUTICALLY ACCEPTABLE COMPOSITIONS COMPRISING ANTIBODY MOLECULES SPECIFIC TO LAMININ-5 α3 CHAIN DOMAINS G1G2 AND USE THEREOF|
Country Status (1)
|WO (1)||WO2008005828A2 (en)|
Cited By (15)
|Publication number||Priority date||Publication date||Assignee||Title|
|EP2198884A1 (en)||2008-12-18||2010-06-23||Centre National de la Recherche Scientifique (CNRS)||Monoclonal antibodies directed against LG4-5 domain of alpha3 chain of human laminin-5|
|JP2012530045A (en) *||2008-06-18||2012-11-29||カール トゥリッグバソン，||Antibody against the domain of laminin -332|
|CN103068847A (en) *||2010-08-24||2013-04-24||罗切格利卡特公司||Activatable bispecific antibodies|
|CN104857511A (en) *||2015-02-13||2015-08-26||浙江大学||Ginsenoside-containing vaccine diluent|
|US9266967B2 (en)||2007-12-21||2016-02-23||Hoffmann-La Roche, Inc.||Bivalent, bispecific antibodies|
|US9382323B2 (en)||2009-04-02||2016-07-05||Roche Glycart Ag||Multispecific antibodies comprising full length antibodies and single chain fab fragments|
|US9676845B2 (en)||2009-06-16||2017-06-13||Hoffmann-La Roche, Inc.||Bispecific antigen binding proteins|
|US9688758B2 (en)||2012-02-10||2017-06-27||Genentech, Inc.||Single-chain antibodies and other heteromultimers|
|US9879095B2 (en)||2010-08-24||2018-01-30||Hoffman-La Roche Inc.||Bispecific antibodies comprising a disulfide stabilized-Fv fragment|
|US9890204B2 (en)||2009-04-07||2018-02-13||Hoffmann-La Roche Inc.||Trivalent, bispecific antibodies|
|US9982036B2 (en)||2011-02-28||2018-05-29||Hoffmann-La Roche Inc.||Dual FC antigen binding proteins|
|US9994646B2 (en)||2009-09-16||2018-06-12||Genentech, Inc.||Coiled coil and/or tether containing protein complexes and uses thereof|
|US10106612B2 (en)||2012-06-27||2018-10-23||Hoffmann-La Roche Inc.||Method for selection and production of tailor-made highly selective and multi-specific targeting entities containing at least two different binding entities and uses thereof|
|US10106600B2 (en)||2010-03-26||2018-10-23||Roche Glycart Ag||Bispecific antibodies|
|US10138293B2 (en)||2007-12-21||2018-11-27||Hoffmann-La Roche, Inc.||Bivalent, bispecific antibodies|
- 2007-06-29 WO PCT/US2007/072457 patent/WO2008005828A2/en active Application Filing
Non-Patent Citations (2)
|HIROSAKI T. ET AL.: 'Structural Requirement of Carboxyl-terminal Globular Domains of Laminin alpha3 Chain for Promotion of Rapid Cell Adhesion and Migration by Laminin-5' THE JOURNAL OF BIOLOGICAL CHEMISTRY vol. 275, no. 29, 21 July 2000, pages 22495 - 22502 *|
|MIZUSHIMA H. ET AL.: 'Identification of Integrin-dependent and -independent Cell Adhesion Domains in COOH-terminal Globular Region in Laminin-5 alpha3 Chain' CELL GROWTH AND DIFFERENTIATION vol. 8, September 1997, pages 979 - 987 *|
Cited By (18)
|Publication number||Priority date||Publication date||Assignee||Title|
|US9266967B2 (en)||2007-12-21||2016-02-23||Hoffmann-La Roche, Inc.||Bivalent, bispecific antibodies|
|US10138293B2 (en)||2007-12-21||2018-11-27||Hoffmann-La Roche, Inc.||Bivalent, bispecific antibodies|
|JP2012530045A (en) *||2008-06-18||2012-11-29||カール トゥリッグバソン，||Antibody against the domain of laminin -332|
|US8545845B2 (en)||2008-06-18||2013-10-01||Karl Tryggvason||Antibodies against domains of laminin-332|
|WO2010070134A1 (en)||2008-12-18||2010-06-24||Centre National De La Recherche Scientifique (Cnrs)||Monoclonal antibodies directed against lg4-5 domain of alpha3 chain of human laminin-5|
|US8431686B2 (en)||2008-12-18||2013-04-30||Centre National De La Recherche Scientifique (Cnrs)||Monoclonal antibodies directed against LG4-5 domain of alpha3 chain of human laminin-5|
|EP2198884A1 (en)||2008-12-18||2010-06-23||Centre National de la Recherche Scientifique (CNRS)||Monoclonal antibodies directed against LG4-5 domain of alpha3 chain of human laminin-5|
|US9382323B2 (en)||2009-04-02||2016-07-05||Roche Glycart Ag||Multispecific antibodies comprising full length antibodies and single chain fab fragments|
|US9890204B2 (en)||2009-04-07||2018-02-13||Hoffmann-La Roche Inc.||Trivalent, bispecific antibodies|
|US9676845B2 (en)||2009-06-16||2017-06-13||Hoffmann-La Roche, Inc.||Bispecific antigen binding proteins|
|US9994646B2 (en)||2009-09-16||2018-06-12||Genentech, Inc.||Coiled coil and/or tether containing protein complexes and uses thereof|
|US10106600B2 (en)||2010-03-26||2018-10-23||Roche Glycart Ag||Bispecific antibodies|
|CN103068847A (en) *||2010-08-24||2013-04-24||罗切格利卡特公司||Activatable bispecific antibodies|
|US9879095B2 (en)||2010-08-24||2018-01-30||Hoffman-La Roche Inc.||Bispecific antibodies comprising a disulfide stabilized-Fv fragment|
|US9982036B2 (en)||2011-02-28||2018-05-29||Hoffmann-La Roche Inc.||Dual FC antigen binding proteins|
|US9688758B2 (en)||2012-02-10||2017-06-27||Genentech, Inc.||Single-chain antibodies and other heteromultimers|
|US10106612B2 (en)||2012-06-27||2018-10-23||Hoffmann-La Roche Inc.||Method for selection and production of tailor-made highly selective and multi-specific targeting entities containing at least two different binding entities and uses thereof|
|CN104857511A (en) *||2015-02-13||2015-08-26||浙江大学||Ginsenoside-containing vaccine diluent|
Also Published As
|Publication number||Publication date|
|AU2008275589B2 (en)||Combination therapies employing GITR binding molecules|
|AU780454B2 (en)||Methods and compositions for modulating cell proliferation and cell death|
|JP5214252B2 (en)||Humanized antibody l243|
|US7276372B2 (en)||Antibodies against GPR64 and uses thereof|
|AU2008338591B2 (en)||Binding molecules to the human OX40 receptor|
|US8598322B2 (en)||Anti CEACAM1 antibodies and methods of using same|
|US9556271B2 (en)||Anti-CEACAM1 recombinant antibodies for cancer therapy|
|EP1575509B1 (en)||Epha2 agonistic monoclonal antibodies and methods of use thereof|
|ES2384168T3 (en)||Methods of Treatment using CTLA-4 antibodies|
|JP5276017B2 (en)||Use of anti egfr antibodies in the treatment of Egfr variant mediated diseases|
|ES2427964T3 (en)||New anti-IGF-IR and its applications|
|EP3409689A1 (en)||Anti-cd134 (ox40) antibodies and uses thereof|
|RU2650869C2 (en)||Antibodies to the related cancer-embryonic antigen cell adhesion molecule (ceacam)|
|CN101014365B (en)||Combination treatment for non-hematologic malignancies using an anti-igf-1r antibody|
|US9573999B2 (en)||CTLA-4 antibody dosage escalation regimens|
|AU2008323206B2 (en)||AXL antibodies|
|ES2595091T3 (en)||Pharmaceutical compositions resistant soluble CEA|
|US20050013819A1 (en)||Use of EphA4 and modulator of EphA4 for diagnosis, treatment and prevention of cancer|
|JP2007509067A (en)||EphA2 vaccines based on Listeria|
|CN105492025A (en)||Methods of treating cancer using pd-1 axis binding antagonists and tigit inhibitors|
|KR20140091040A (en)||Antibodies specific for trop-2 and their uses|
|Gordon et al.||CA125-and tumor-specific T-cell responses correlate with prolonged survival in oregovomab-treated recurrent ovarian cancer patients|
|AU2011255870B2 (en)||Anti-human TROP-2 antibody having antitumor activity in vivo|
|JP4854912B2 (en)||Antibodies against cancer|
|AU2001267780B2 (en)||Therapeutic method and composition utilizing antigen-antibody complexation and presentation by dendritic cells|
|121||Ep: the epo has been informed by wipo that ep was designated in this application||
Ref document number: 07799175
Country of ref document: EP
Kind code of ref document: A2
|NENP||Non-entry into the national phase in:||
Ref country code: DE
|NENP||Non-entry into the national phase in:||
Ref country code: RU
|122||Ep: pct application non-entry in european phase||
Ref document number: 07799175
Country of ref document: EP
Kind code of ref document: A2