WO2016133829A1 - Nouveaux composés de liaison à sd1 et leurs utilisations - Google Patents

Nouveaux composés de liaison à sd1 et leurs utilisations Download PDF

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Publication number
WO2016133829A1
WO2016133829A1 PCT/US2016/017888 US2016017888W WO2016133829A1 WO 2016133829 A1 WO2016133829 A1 WO 2016133829A1 US 2016017888 W US2016017888 W US 2016017888W WO 2016133829 A1 WO2016133829 A1 WO 2016133829A1
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seq
antibody
antibody binding
binding
isolated antibody
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PCT/US2016/017888
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English (en)
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Robert B. Dubridge
Veronica Juan
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Full Spectrum Genetics, Inc.
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Publication of WO2016133829A1 publication Critical patent/WO2016133829A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]

Definitions

  • PHHH Mesothelin is. GP!-iiaked protein which is over-expressed, OH the surface of a. variety of human cancer ceils (pancreatic cancer, mesothelioma, ovarian cancer, prostate cancer, breast cancer,
  • cinolaagiocarciaoma squamous cell carcinoma aod !uag adenocarcinoma
  • a lower level expression of the protein is restricted w the mesothelium in normal adult tissues.
  • mesothelin represents an attractive therapeutic target for imas ROtherapy.
  • PKH)2j SDl is a human, single domain antibody (heavy chain only) isolated from a synthetic phage library. SDl inds specifically to domain ⁇ of mesothelin and has strong ADCC and CDC activity in vitro against tumor est! lines overexpressing the mesothelin protein JMol Cancer Ther 12:416-26 ⁇ 20!3)
  • the present invention is directed to novel SDl antibody binding compounds and methods of using the same, Aspects and embodiments of the present invention are exemplified in a number of implementations and applications, some of which are summarized below and throughout the specification.
  • the invention is directed to novel antibody binding compounds specific for the human mesotheiin target molecule, w erein suds antibody binding compounds comprise complementary determining regions defined by the following amino acid sequences:
  • X: is S, A or G
  • X. ⁇ is H, f or Y;
  • Xs is Y or Q
  • i is A or P; and wherein ⁇ !( 3 ⁇ 4 X ⁇ , ,, X s , X X ? , X*, X 3 ⁇ 4 Xw nd X is are not smmhaneousiy D, ,E, S, H, G, T, D, Y, L, R, and A, respectively.
  • lite single capital letters are the standard SUP AC codes for amino acids.
  • such antibody binding compounds have an affinity for the raesothelin target molecule that is characterized fay an equilibrium binding constant of 100 nM or less, as measured by conventional techniques,
  • compounds of the invention comprise the polypeptide of SEQ ID NO: 4 with one or more of the following amino acid substKutions: D28L, D28K, D28Q, D28H, D28A, D28.E, D28I, E33I, E33D, S35A, S35G, H53F, H53Y, G55S. G55A, G55E, T61D, D62R, D62 , Y95Q, L97i, R9S , R98L, and A10I P.
  • compounds of the invention comprise the polypeptide of SEQ ID NO: 4 with one or more of the following amino acid substitutions: D28L, D28K, D28Q, D28H, D28A, D28E, D28I, £31, E33D, S35A, S3SG, M53E, B53Y, G55S, G55.A, G55E, T61D, D62R, D62K, Y95Q, L97i, R98K, R98L, and A 10 IP, wherein at least one of the one or more amino acid substitutions is non-conservative, in some embodiments, where the framework residues are those of SEQ ID NO: 4, or at.
  • compounds of the invention comprise the polypeptide of SEQ ID NO: 4 with one or more of the following amino acid substitutions: D28L, D.28 , D28Q, D28M, D28A, JD28I, E.331, S35G, H53F, H53Y, G55S, GS5A, G55E, T61 , D62R, D62K, Y95Q, R98.L, and A ! 01 P.
  • the foregoing antibody binding compounds each have aa affinity for She raesothelin target molecule that is characterised by an equilibrium binding constant of 100 nM or less, as measured by conventioaal techniques.
  • compounds of the invention comprise the polypeptide of SEQ ID NO: 4 with at least two of the following amino acid, substitutions: D28L, D28K, D28Q, D28I1 D28A, D2SE, D28I, E33I, E33.D, S3SA, S35G, H53F.
  • compounds of the invention comprise she polypeptide of SEQ ID NO: 4 with at least two of the following amino acid substitutions: D28L, D28 , D28Q, D28H, D28A, D2SE, D2Si, E33I, E33D, S35A, S35G, HS3F, H53Y, G55S, G55A, G55E, T61D, D62R, D62 , Y95Q, L97I, R98K, R98L, and A101P, wherein at least one of the one or more amino acid substitutions is non-eonssrvative.
  • the foregoing antibody binding compounds each have an affinity for the mesofhe!in target molecule that is characterized by an equilibrium binding constant of 100 tiM or less, as measured by con v eiit tonal teclm iq ues .
  • antibody binding compounds of the invention comprise a polypeptide defined by the following formula:
  • rt, 3 ⁇ 4 and 3 ⁇ 4 are CDR regions of SEQ ID NO: I , SEQ ID NO; 2, and SEQ ID NO: 3, respectively, as described, above, and. wherein f i? ⁇ 6 and. ft axe framework residues selected so that the compound of equation (1) is at !east eighty percent identical to the poiypeptide of SEQ ID NO: 4, in.
  • A, i3 ⁇ 4 ft and ft are framework residues selected so that the compound of equation 0) is at least ninety percent identical to the polypeptide of SEQ ID NO: 4; and in still other embodiments, ft, ft, f ⁇ and ft are framework residues selected so that the compound of equation (I) is at least ninety-five percent identical to the polypeptide of SEQ ID NO: 4.
  • the antibody binding compounds of equation (!) each have an affinity for the inesothelin target molecule that is characterized by an equilibrium binding constant of 100 nM. or less, as measured by conventional fechnlqnes.
  • an antibody binding compound of the invention (sometimes referred to herein as "08") comprises a polypeptide wherein j, t3 ⁇ 4 and r.$ are as follows:
  • a D& antibody binding compound of the invention comprises a polypeptide defined by equation (I) wherein ft, f3 ⁇ 4 % and .ft are as described above.
  • Some antibody binding compounds of this embodiment in particular comprises the polypeptide of SEQ ID NO: 5,
  • an antibody binding compound of the invention (sometimes referred to herein as "H?") comprises a polypeptide wherein n, 3 ⁇ 4 and r3 ⁇ 4 are as follows :
  • an H7 antibody binding compound of fee invention comprises a poiypeptide defined by equation ( ⁇ ) wherein ft, ft, ft and ft are as described above.
  • Some antibody binding compounds of this embodiment in particular comprises the polypeptide of SEQ ID NO: 9.
  • an antibody binding compound of the invention (sometimes referred to herein as 3 ⁇ 4 B:l i") comprises a polypeptide wherein n, n and. r? are as follows: irt
  • a B l 1 antibody binding compound of the invention comprises a polypeptide defined by equation 0.) wherein ft., f 3 ⁇ 4 % and .3 ⁇ 4 are as described above.
  • Some antibody binding compounds of this embodiment in particular comprises ihe polypeptide of SEQ ID NO: 13.
  • an antibody binding compound of the invention (sometimes referred to herein as T12" ⁇ comprises a polypeptide wherein rj, r> and r? are as follows:
  • an F 12 antibody binding compound of the invention comprises a polypeptide defined by equation (I) wherein f>, f 3 ⁇ 4 3 ⁇ 4 and 3 ⁇ 4 are as described above.
  • Some antibody binding compounds of this embodiment in particular comprises the polypeptide of SEQ ID NO: 1?.
  • ait aiitibody binding, compound of the invention (sometimes referred to herein as "G12") comprises a polypeptide wherei n, r3 ⁇ 4 aad r? are as follows:
  • a 012 antibody binding .compound of the mvention comprises a polypeptide de fined by equation ⁇ 1 ⁇ wherein ft, f3 ⁇ 4 f3 and f ⁇ are as described above.
  • Some antibody binding compounds of this embodiment in particular comprises the polypeptide of SEQ ID NO: 21.
  • an aiitibody binding compound of the invention (sometimes referred to herein as "4C") comprises a polypeptide of a form defined by Equation (I) wherein n, r? and t . are as follows:
  • a 4C antibody binding compound of the invention comprises a polypeptide defined by equation ( ⁇ ) wherein f Js f3 ⁇ 4 3 ⁇ 4 and .3 ⁇ 4 are as described above,
  • ft, fc, ft and 3 ⁇ 4 of a 4C antibody binding compound axe the framework regions of SEQ ID NO: 4,
  • an antibody binding compound of the invention (sometimes referred to herein as ' ⁇ ) comprises a .polypeptide of a form defined by Equation (I) wherein n , r and r$ are as follows:
  • a 1H antibody binding compound of the invention comprises a polypeptide defined by equation (I.) wherein f3 ⁇ 4 3 ⁇ 4 and ⁇ £» are as described ' above, in. some embodiisents, S s ⁇ , ⁇ and & of a i.H antibody binding compound are the framework regions of SEQ ID NO: 4.
  • an antibody binding compound of the invention (sometimes referred to herein as "2G") comprises a polypeptide of a form defined by Equation (I.) wherein rt, 3 ⁇ 4 and rj are as follows: in. still another embodiment, a 2G antibody binding compound of the invention comprises a polypeptide defined by equation ( ⁇ ) wherein, fj. f? ( fj and 3 ⁇ 4 are as described above. In. some embodiments, i , 3 ⁇ 4, 3 ⁇ 4 and of a 2G antibody binding compound are the framework regions of SEQ ID NO: 4.
  • the above antibody binding compounds of the invention are each selected with an afiluiry for the mesothciiu target molecule that is characterized by an equilibrium binding constant of 1 0 nM or less, as measured by conventional techniques; or 10 nM or less, as measured by conventional techniques; or i n or less, as measnred by conventional techniques.
  • the invention also includes pharmaceutical compositions comprising at least one of the .foregoing antibody binding compounds.
  • the invention fuitber provides the use of an an tibody binding compound of the invention in the preparation of a medicament for the therapeutic and/or
  • compositions of the invention comprise one or more antibody binding compounds of the invention and a carrier, in one embodiment, the carrier is pbarmaeeuticaiiy acceptable.
  • the invention provides nucleic acids encoding an antibody binding compound of the iavemsoa. in some erabodiments of this aspect a nucieic acid cneodiag an antibody binding compound of tlie invention is an isolated nucieic acid.
  • the invention provides vectors comprising a nucleic acid of the invention.
  • the invention provides host cells comprising a nucleic acid or a vector of the invention.
  • a vector can be of any type, for example, a recombinant vector such as an expression vector. Any of a variety of host ceils can be used.
  • a host cell is a prokaryotic cell, for example, E, coii.
  • a host ceil is a eukaryotic ceil, for example a mammalian ceil such as Chinese Hamster Ovary (CHO) cell.
  • the invention provides methods of making an antibody binding compound of the in vention.
  • the in vention provides methods of making an antibody of the inven tion (which, as defined herein includes Ml length antibody and fragments thereof), said method comprising expressing in a suitable host cell a recombinant vector of the invention encoding the antibody (or fragment thereof), and recovering the antibody.
  • the invention provides an article of manufacture comprising a container; and a composition contained within the container, wherein the composition comprises one or more antibody binding compounds of the invention.
  • the composition comprises a nucleic acid of the invention
  • a composition comprising an antibody further comprises a carrier, which in some embodiments is pharmaceutically acceptable
  • an article of manufacture of the invention farther: comprises instructions for adn ustering the composition (e.g., the antibody ) to ait individual (such as instructions for any of the methods described herein).
  • the invention provides a kit comprising a first container comprising a composition comprising one or more antibody binding compounds of the isiveaiioii; and a second container comprising a buffer, in one embodiment, the buffer is pharmaceutically acceptable.
  • a composition comprising an antibody binding compound further comprises a carrier, which in some embodiments is pharmaceutically acceptable.
  • a kit Farther comprises instructions for administering the composition (e.g., the antibody) to an individual.
  • the invention provides use of an antibody binding compound of the invention in the preparation of a medicament for the therapeutic and/or prophylac tic treatment of a disorder, such as a cancer, a tumor, and or a cell, proliferative disorder.
  • a disorder such as a cancer, a tumor, and or a cell, proliferative disorder.
  • the invention provides a use of an. antibody binding compound of the invention, as a component of a cell-based therapeutic, such as a CAR of an engineered T lymphocyte.
  • Fig. lA illustrates binding- titration data, for antibody- inding compounds of the invention.
  • Fig. IB is a table showing binding data of antibody blading compounds of the invention.
  • the practice of the present invention may employ, unless otherwise indicated, conventional techniques of organic chemistry, molecular biology, ceil biology, biochemistry, and therapeutic antibody development, which are within the skill of the art Specific ilwstratioas of suitable techniques can be had by reference to the examples below, with the understanding that other equivalent techniques and procedures can be used.
  • Conventional techniques and guidance related to making and using the invention may be found in standard treatises and laboratory manuals, such as Genome Analysis: A laboratory Manual Series (Vols, i- IV); PCR Primer; A laboratory Manual; Phage Display; A laboratory Manual; ami Molecular Cloning: A. Laboratory Mamtal (all from Cold Spring Harbor .Laboratory Press); Sidhu, editor, Phage Display in Biotechnology and Drug Discover?
  • An antibody binding compound of the invention may be produced and/or used in a variety of formats, including but not limited to, a monoclonal antibody or Fc fusion protein, a monoclonal antibody or Fc fusion protein of a selected isotype, an antibody frapnent, a humanized monoclonal antibody or Fc fusion protein, a glycosylated monoclonal antibody or Fc fusion protein, an antibody or Fc fusion protein conjugated to another moiety that imparts an added functionality, e.g, cytotoxicity, so the resulting conjugate, and the like.
  • a particular format may depend on a variety of factors, including but not limited, to, tissue accessibility, whether ADCC is desired, solubility, whether bi-specificity is desired, ease of raanulacture, and the like. It is understood that in some embodiments, compounds of Site invention may be paired with other antibody binding regions, e.g. from a light chain, to form a multi-chain, e.g. a two-chain, antibody or antibody fragment.
  • the present invention encompasses antibody fragments.
  • antibody fragments In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access Co solid, tumors.
  • Various techniques are available for the production of antibody fragments.
  • Antibody .fragments may be derived via proteolytic digestion of intact antibodies (sec, e.g. , Moriinoto et al., Journal of Biochemical and Biophysical Methods 24: 107- ⁇ 17 ( 1992); and Breunan et at,. Science, ' 229:81 (1985)).
  • Antibody fragments may also be produced directly by recombinant host cells. ScFv antibody fragments can be expressed in and secreted from £.
  • Antibody fragments can be isolated from the antibody phage libraries discussed above.
  • Fab'-SH fragments can be directly recovered from E. coii and chemically coupled to form F ⁇ a ' )2 fragments (Carter et aL, Bio/Tedmology 10: 163-16? (1 92)).
  • F(ab')2 fragments can be isolated directly from .recombinant ' host ' cell culture.
  • Fab and Ffab S fragment with increased in ivo halt-life comprising a Salvage receptor binding epitope residues are described in U.S. Pat. Mo. 5,869,046.
  • the antibody of choice is a single chain Fv fragment (scFv) (see, e.g., WO 93. 16185; US. Pat. os. 5,571,894 and 5,587,458).
  • Fv and sFv are the only species with intact combining site that are devoid of constant regions; thus, they are suitable for reduced nonspecific binding daring in vivo use.
  • sFv fusion proteins may be constructed to yield, fusion of an effector protein at either the amino or the carbosy terminus of an sFv. See Antibody Engineering, ed.
  • the antibody fragment niay also be a 'linear antibody," e.g., as described, for example, in U.S. Pat. Mo. 5,641 ,870.
  • Such linear antibody fragments may he
  • Humanized antibodies of the invention include those that lave amino acid substitutions in the framework region (PR) and affinity tnaiuratioii variants wi th changes in the grafted CD s.
  • the antibodies of the in vention further comprise changes in amino acid residues in the Fe region that lead to improved effector function including enhanced complement dependent cytotoxicity (CDC • and/or antibody dependent cellular cytotoxicity (ASDCC) function and B-ceil killing.
  • Other antibodies of the invention include those having specific changes that improve stability, in some embodiments, the antibodies of the invention are of the IgG c lass (e.g., IgG! o Ig04),
  • bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities lor at least two different antigens. In the present case, one of the binding specificities is for antigen of the inventive compounds and the other is for any other antigen.
  • Bispecific antibodies may also be used io localize cytotoxic agents to ceils which express the antigen of the inventive compounds. These antibodies possess an arm binding to the inventive compound antigen and an arm whic binds the cytotoxic agent (e.g., saporin, anii-mterferon- ⁇ , vines alkaloid, richi A chain, methotrexate or radioactive isotope hapten). Bispeeifie antibodies may also be used to localize cytotoxic celts to cells which express the antigen of the inventive compound (atrti-CIB, anti-CD 16, MieA, etc).
  • cytotoxic agent e.g., saporin, expressing-mterferon- ⁇ , vines alkaloid, richi A chain, methotrexate or radioactive isotope hapten.
  • Bispeeifie antibodies may also be used to localize cytotoxic celts to cells which express the antigen of the inventive compound (atrti-CIB, anti-CD 16, MieA, etc).
  • Bispeei fie antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab')2 bispeeifie antibodies, scFv fusions or the like). Methods fee tasking bispeeifie antibodies are known in the art Traditionally, the recombinant production offoispecific antibodies is based on the co-expression of two inisntmoglobutin heavy chaiir ight chain pairs, -where th two heavy chains have different specificities (Miistem and Cuello, Nature, 305: 537 (1983)). Similar procedures are disclosed in WO 93/08829 published May 13, 1993, and in
  • antibody variable domains with the desired binding specificities ⁇ antibody- antigen combining sites are fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first .heavy-chain constant region (CH I ), containing the site necessary for light chain blading, present in at least one of the fusions.
  • DNAs encoding the instBanogiobulin heav -chain fusions and, if desired, the immuiwglob tin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
  • the bispeeifie antibodies are composed of a hybrid inimnooglobalin 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.
  • ft was found that this asymmetric structure facilitates the separation of the desired bispeei fie compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulm light chain in only one hal f of the bispeeifie molecule provides for a facile way of separation, This approach is disclosed in WO 94/04690.
  • For farther details of generating bispeeifie antibodies see, for example, Suresh et at. Methods in Enzytnology, ! 2i :2 H) (1986).
  • Antibody binding compounds of the invention may be glycosylated. Addition of glycosylaiion sites to an antibody binding compound is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the tripeptide sequences for N-linked gtycosy latiou sites, or so that it contains one or more serine or threonine residues to the sequence of the original antibody for O-linfced glycosySatiott sites,
  • the carbohydrate attached thereto may be altered.
  • antibodies wish a mature carbohydrate structure that lacks fucose attached to an Fc region of the antibody are described in US Pat Appl No US 2003/0157108 (Presta, L,). See also US 2004/009362 J ( yowa Hakko J ogyo Co., Ltd).
  • Antibodies with a bisecting N-acetylglucosam»ne (GlcNAe) in the carbohydrate attached to an Fc region of the antibody are referenced in WO 2003/01 i 878, Jean- airet et al. and U.S. Pat, No. 6,602,684, Uroaua et ai.
  • oligosaccharide attached to an Fc region of the antibody are reported hi WO 1997/30087, Patel et al. See, also, WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.) eoree ing antibodies with altered carootrydtate attached to theFc region, thereof; See also US 2005/0123546 (Umaaa et al) on aMigen-binding molecules with modified giycosylation.
  • the Fc region further comprises one or more amino acid substitutions therein which further improve ADCC, for example, substitutions at positions 298, 333, aid/or 334 of the Fc region (Ho numbering of residues).
  • Examples of publications related to w defucosylated w or "fucose-deficienr antibodies include: US 2003/0157508; WO 2000/61739; WO 2001/29246; US 2003/01 15614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US
  • ceil li nes ptoducing . defucosyiaied antibodies include Lecl 3 CHO cells deficient ia protein fucos httion (lipka et al Arch. Biochetn, Biophys.
  • antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an iminunoglobulin heavy chain constant domain, comprising at least part of the hinge, CR2, and CI-13 regions, it is preferred to have the first heavy-chain constant region (CHI ' ), containing the site necessary for light chain binding, present iu at least one of the fusions.
  • DMAs encoding she immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
  • Antibody binding compounds of the present invention ca be further modified to conta in addit ional nonproteinaceous moieties that are known in the art and readily available.
  • the moieties suitable for derivatization of the antibody binding compounds are water soluble polymers.
  • water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose.
  • dextran polyvinyl alcohol, polyvinyl pyrrolidone, poly- 1 ,3-dioxo.lane, poly-l,3,6-trioxane, ethylcnc/maieic anhydride copolymer, poiyatninoaeids (either homopolyrners or random copolymers), and dextrin or po!y( «-vm l Dyn'o o «e)polyeShyiene glycol, propropyiene glycol honwpolvmers, polypropylene oxide ed3 ⁇ 4y1ei3 ⁇ 4e oxide co-polymers, polyoxyerhylated poiyois (e.g., glycerol), polyvinyl alcohol, aad mixtures thereof.
  • Polyethylene glycol propionaidehyde nay have advantages " m rnanufecluring dye to its stability " m water.
  • the polymer (Hay be of. any molecular weight * and stay be branched or unbraached.
  • the auraber of polymers attached to the antibody may vary, aad if more than one polymers are attached, they can. be ie same or different molecules, i general, the number and/or type of polymers used For derivatizaiion can be determined based oa considerations including, but not limited to, the particular properties or fimetions of the antibody to be improved, whether t he antibody derivative will be used in therapy under defined conditions, etc.
  • Antibody binding compounds of she invention ma include chimeric antigen receptor compounds (CARs), whenever a particular antigen serves as a target for cytotoxic T cells * such as a tumor antigen.
  • C ARs permit MHC-ittdependent targeting of cytotoxic T cells aad are made by fusing antigea-bindiiig components of the invention with transmembrane and signaling components capable of eliciting a cytotoxic T cell s nse henever the- antigen-specific Component balding to its target.
  • Exemplary re ferences teaching the manufacture aad application of CARs include the following references thai are incorpora ted herein by reference: U.S.
  • the invention includes chimeric antigen receptor compounds comprising at least, one transmembrane polypeptide comprising at least, one extracellular iigaisd-biiidiag domain aad one transmembrane polypeptide comprising at least one signai-tftnisducmg domain; wherein the polypeptides assemble together to form a niulti-ehain CAR, and wherein the at least one extracellular Hgan.d-bin.ding domain comprises aa antibod binding compound of the invention, fit some embodiments, the invention includes an isolated immune cell comprising at least one such chimeric antigen receptor compounds. In. some embodiments, such immune cell may comprise a T-tympiiocyte,
  • the nucleic acid encoding it is isolated and inserted into a replicabie vector for further cloning (amplification of the DN ) or for expression.
  • DNA encoding the antibody is readily isolated and sequenced using conventional procedures. Marty vectors are available. The choice of vector depends in part on the host ceil to be used. Generally, preferred, host cells are of either prokaryotic or eukaryotic (generally mammalian) origin, ft will be appreciated that constant regions of any isotype caa be used for this purpose, including IgG, Ig , IgA, Ig , aad IgE constant regions, and that such constant regions can be obtained from any human or animal species.
  • Poiyrtucleotide-sequeuces encoding polypeptide components of the antibody of the invention can be obtained using standard recombinant techniques. Desired, polynucleotide sequences may be isolated and sequenced from antibody producing ceils such as hybridoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding She polypeptides are inserted into a recombinant vector capable of rep licating and expressi ng heterologous polynucleotides in prokarycuic hosts. Many vectors that are available and known in the art can be used for the purpose of the present invention.
  • Selecti n of an appropriate vector will depend mainly one the s.i3 ⁇ 4e of the nucleic acids to be insetted iato the vector and the particular host cell to be transformed with the vector.
  • Each vector contains various components, depending en its Junction .(amplification or express ion of heterologous polynucleotide, or both ⁇ and. its ' compatibility with the particular host cell in which is resides.
  • the vector components generally include; but are not limited to; an origin of replication, a selection marker gene, a promoter, ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid, insert and a transcription termination sequence.
  • ptasnrid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts.
  • the vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells.
  • E. coli is typically transformed using pBR322, a plasrai derived from an E. coli species.
  • pB .322 contains genes encoding ampfciiiin (Amp) and tetracaine (let) resistance and tints provides easy means for identifying transformed ceils, p.B1322 s .
  • microbial piasniid or bacteriophage may also contain, or can be modified to contain, promoters which can be used by the .microbial organism for expression of endogenous proteins.
  • promoters which can be used by the .microbial organism for expression of endogenous proteins.
  • pBR322 derivatives used for expression of particular antibodies are described in detail in Carter et a!,,. U.S. Pat. No, 5,648,237,
  • phage vectors containing replico and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts.
  • bacteriophage such as GEMTM-:i I may be utilized in inafciiig a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.
  • the expression vector of the invention may comprise two or more promoter-cistron pairs, encoding each of the polypeptide components.
  • a promoter is an retranslated regulatory sequence located upstream ($') to a eistron that modulates its expression.
  • Prokaryotie promoters typically fall ' into two ' classes, inducible and constitutive.
  • Inducible promoter is a promoter that initiates increased levels of transcription of the eistron under its control iu response to changes in the culture condition, e.g., the presence or absence of a nutrient or a change in. tearpenaure.
  • the selected promoter can be operabiy linked to eistron DMA encoding a heav chain by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated, promoter sequence into the vector of the invention.
  • Both the native promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of the target genes, la some embodiments, heterologous promoters are utilized, as they generally permit greater transcription and higher yields of expressed target gene as compared to trie native target polypeptide promoter.
  • Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the ⁇ -gaiactanase and lactose promoter systems, a tryptophan (ttp) promoter system and hybrid promoters suc as the tac or the trc promoter.
  • ttp tryptophan
  • Other promoters that are functional in bacteria are suitable as well.
  • Their nucleotide sequences have been, published, thereby enabling a skilled worker operabiy to ligate them to eistrons encoding the target light and heavy chains (Siebenlisi et aL (i 980 ⁇ Cell 2.0: 2.69 ⁇ using tinkers or adaptors to supply any required restriction sites,
  • each cistmn within the recombinant vector comprises a secretion signal sequence component that directs translocation, of the expressed polypeptides across a membrane.
  • the signal sequence may be a component of the vector, or it may be a part of the targe t polypeptide D A that is inserted into die vector.
  • the signal sequence selected for (fee purpose of this invention should be one that is recognised and processed (i.e., cleaved b it signal peptidase) by the host cell.
  • the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group consisting of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STil) leaders, La aB, PhoE, PelB, OmpA, and MBP,
  • STil enterotoxin II
  • the signal sequences used in both eistrons of the expression system are ST1I signal sequences or variants thereof.
  • the production of the immunoglobulins according to the invention can occur in the cytoplasm of the host cell, and therefore does not require the presence of secretion signal sequences wi thin each cistron, in that regard, immunoglobulin light and heavy chains are expressed, folded and assembled to form functional immunoglobulins within the cytoplasm.
  • Certain host strains e.g., the E, coli fcrxB-strams
  • Prokaryotic host ceil satiable for expressing antibodies of the invention include Archaebscferia and Eubacferia, such as Gram-negative or Grain-positive organisms.
  • useful bacteria include Escherichia (e.g., E, coli). Bacilli (c.g.,B. snbtilis), Enterobacteria, Pseudomortas species (e.g., P.
  • Salmonella typhimtiriuin, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhfeobia,
  • E. coli ceils are used as hosts for the invention.
  • E, coli strains include strain. W3 ! 10 (Baehmann, Cellular and Molecular Biology, vol. 2 (Washington, D. : American Society for Microbiology, 1987), pp, 1 190-1219; ATCC Deposit No. 27,325) and derivatives thereof; including strain 33D3 having genotype W3 I 1 AihuA ( ⁇ ) ptr3 lac Iq lacL8 AompTACnrapc-fepE) degP41 katiR (U.S. Pat. No. 5,639,635).
  • E. coli 294 ATCC 31 ,446
  • E. coli 8, E, col& 1776 ATCC 3 ! ,537)
  • E, coli RV308 ATCC 1,608
  • E. coli 294 ATCC 31 ,446
  • E. coli 8, E, col& 1776 ATCC 3 ! ,537
  • E, coli RV308 ATCC 1,608
  • coli, Sesxaiia, or Salmonella species can be suitably used as the host when well-known plasmtds such as pBR322, pBR32S, pACYCl ' 77, or pKN410 are used to supply the rep!iec-n.
  • plasmtds such as pBR322, pBR32S, pACYCl ' 77, or pKN410 are used to supply the rep!iec-n.
  • the host cell should secrete mmimal amounts of proteolytic enzymes, and additional protease inhibitors rnay desirabl be incorporated hi the cell culture.
  • Host cells are . transformed wifit the above-described expression vectors and cultured in conventional nutrient media modified as appropriate For inducing promoters, selecting irarisformants, or amplifying tbe genes encoding she desired sequences.
  • Transformation means introducing DNA. into the prokaryotic host, so that, the DNA is replicable, either as an extrachtwsosoinat element or by chromosomal integrant, Depending on the host cell used, traasfonnauou is done using standard techniques appropriate to such cells.
  • the cai um treatment employing calcium chloride is generally used for bacterial cells that contain substantial cell-wall barriers.
  • Another method for transfonaation employs polyethylene glycol/DMSO.
  • Yet another technique used is
  • Prokaryotic cells used to produce the poiypeptides of the invention are grown m media known in the art and suitable for culture of the selected host cells.
  • suitable media include Luria broth (LB) plus necessary nutrient supplements.
  • the media also contains a selection agent, chosen based on the construction of the expression vector, to selectively permit growth of prokaryotic ceils containing the expression vector. For example, anipicitliii is added to media for growth of cells expressing arapieiltm resistant gene.
  • any necessary supplements besides carbon, nitrogen, and inorganic phosphate sources may also be included at appropriate concentrations introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source.
  • the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cysfaniine, fhiogiyeoilafe, dilhioerythritoi and dirhiothre ol.
  • the prokaryotic host cells are cultured at suitable temperatures.
  • the preferred temperature ranges from about 20* C. to about 39° C, more preferably from about 25" C. to about 37° C, even more preferably at about W C.
  • the pH of the medium may be any pH rangin from about 5 to about 9, depending mainly on the host organism.
  • the pH is preferably from about 6,8 to. about 7.4, and more preferably about 7,0,
  • an inducible promoter is used in the expression vector of the invention, protein expression is induced under conditions suitable for the activation of the promoter.
  • PhoA. promoters are used for controlling transcription of the polypeptides.
  • the transformed host cells are cultured in a phosphate-limiting medium for induction.
  • the phosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons et al., J. Immunol. Methods (2002), 263; 133-1 7).
  • a variety of other inducers may be used, according to the vector construct employed, as is known in the art.
  • the expressed polypeptides of the present invention are secreted into and recovered from the periplasm of the host cells. Protein recovery typically involves disrupting the microorganism, generally by such means as osmotic shock, sosieation or lysis. Once carrotis are disrupted, cell debris or whole ceils may be removed by centnfugation or filtration. The proteins may be further purified, for example, by afiioiiy resin chromatography. Alternatively, proteins can be transported into the eai rre media and isolated therein. Ceiis may be removed from the culture and fee culture supernatant being filtered and concentrated for further purification of the proteins produced. The expressed polypeptides can be further isolated and identified using commonly known methods such as po!yacry!amide ge electrophoresis (PAGE) and Western blot assay,
  • PAGE po!yacry!amide ge electrophoresis
  • antibody production is conducted in large quanti ty by a fermentation, process.
  • Various large-scale fed-batch fermentation procedures are available for production of recombinant proteins.
  • Large-scale fermentations have at least 1000 liter of capacity, preferably about 1,000 to 100,000 liters of capacity. These fermentors use agitator impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon/energy source).
  • Small scale fermentation refers generally to fermentation in a fernientor that is so more than approximately 100 liters in volumetric capacity, and can range from about 1 liter to about 100 liters,
  • induction of protein expression is typically ini tiated after the ceils have been grown under suitable conditions to a desired density, e.g., an OD550 of about 180-220, at. which stage the cells are in the early stationary phase.
  • a variet of inducers may be used, according to the vector construct employed, as is known in the art and described above.
  • Ceils may be grown for shorter periods prior to induction. Ceiis are usually induced for about 12-50 hours, although longer or shorter induction time ma be used,
  • additional vectors oversxpressing chaperone proteins such as Dsb proteins (DsbA, DsbB, DsbC. DsbD, and/or DshG) or FkpA (a pepf ylpro!yl cis,trans-isomerase with chaperone acti ity) can be used to co-transform the host prokaryotfc DCis.
  • Dsb proteins DsbA, DsbB, DsbC. DsbD, and/or DshG
  • FkpA a pepf ylpro!yl cis,trans-isomerase with chaperone acti ity
  • the chaperone proteins have been demonstrated to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al., (1999) I Biol. Chem.
  • host cell strains may be modified to effect genetic nnUationCs) in die genes encoding kn wn bacteria! proteases such «3 ⁇ 4 Protease 10, OtnpT, DegFVTsp, Protease I, Protease Mi, Protease V, .Protease VI, and combinations thereof.
  • proteases such «3 ⁇ 4 Protease 10, OtnpT, DegFVTsp, Protease I, Protease Mi, Protease V, .Protease VI, and combinations thereof.
  • Some E. co!i protease-deficient.straias are available and described in, for example, My et at, (199 supra; Georgiou et at, U.S. Pat. No. 5,264,365; Georgiou ei at, U.S. Pat. No. 5308,192; Hara et at, Microbial Drag ' Resistance, 2:63-72 (1996),
  • Standard protein psmfication methods kftowsr in die art Can be employed.
  • the following procedures are exemplary of suitable purification procedures: fractionation on imm noaifmity or ion-exchange columns, ethanol precipitation, reverse phase MPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocasing, SDS-PAGE, aninionium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.
  • Protein A mitnobiiked on a solid phase is used for iramitnoafilnity purification of the foil length antibody products of the invention.
  • Protein A is a 41 kD ceil wall protein from Staphylococcus aureas which binds with a high a iu ' ty to the Fc region of antibodies, Laidmark et aL (1983) J . Immunol. Metii. 62: 1 -13.
  • the soiid phase to which Protein A is immobilized is preferably a column comprising a glass or silica surface, more preferably a controlled pore glass column or a silicic acid column. In some applications, the column has been coated with a reagent, such as glycerol, in an attempt to prevent nonspecific adherence of contaminants.
  • PK 64j
  • the preparation derived from die ceil culture as described above is applied onto the Protein A immobi lized solid phase to allow specific binding of the antibody of interest to Protein A.
  • the solid phase is then, washed to remove contaminants non-speeifreaiiy bound to the solid phase.
  • the antibody of interest is recovered from the solid phase by elation,
  • Vector components for eukaryotic expression generally include, but are not limited to, one or snore of the following: a signal sequence, an origin of replication, one or snore marker genes, an. enhancer element, a promoter, and a transcription termination sequence.
  • a vector for use in a eukaryotic host cell may also contain a signal sequence o other polypeptide having a specific cleavage site at the N-ieraunus of She mature protein or polypeptide of interest.
  • the heterologous signal sequence selected preferably is one that is recognized and processed (i.e... cleaved by a signal peptidase) by the host, cell.
  • mammalian signal sequences as well as viral secretory leaders for example, the herpes simplex gD signal, are available..
  • the DNA encoding a signal sequence is ligaied in reading frame to DMA encoding the antibody.
  • Origin of Replication Generally, an origin of replication component is not needed for mammalian expression, vectors. For example, the SV40 origin may typically be used only because it contains the early promoter,
  • Selection Gene Component Expression and cloning vectors may contain, a selection gene, also termed a selectable marker.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicilim, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, where relevant, or (c) supply critical nutrients not available from complex media.
  • a selection scheme utilizes a drug to arrest growth, of a host cell Those celts that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and tints survive the selec tion regimen, Examples of such dominant selection use the drugs neomycin, mycophenoiic acid and hygroniycin.
  • suitable seleciabie marker for mammalian cells are those that enable the identification of cells competent to take up the antibody nucleic acid such as DHFR, thymidine kinase, etaUotro nem-1 and -II, preferably primate metailothionein genes, adenosine deaminase, ornithine decarboxylase, etc.
  • cells transformed with the DHFR Selection gene are first identified by cultttritig ail of the transibrm&ots in a culture medium that contains methotrexate (Mix), a competitive antagonist of DHFR.
  • Mc methotrexate
  • An appropriate host cell when wild-type .DHFR is employed- is the Chinese hamster ovary (HO) cell line deficient In DHFR activity ⁇ e.g., ATCC CRt ⁇ 903 ⁇ 4 .
  • host cells particularly wild-type hosts that contain endogenous DHFR transformed or co-transformed with D seq ences encoding- an antibody, wild-type DHFR protein., and. another selectable- marker such as
  • aminoglycoside . ⁇ '-phosphotransferase ⁇ APHI can be selected by cell growth in medium containing a selection agent for the seleciabie marker such as art anitnoglycosidic antibiotic, e.g., kanamycin, neomycin, or 0418. See U.S. Pat, No. 4,965, 199.
  • Promoter Component Expression and cloning vectors usually contain a promoter thai is recognised by the host organism and is operably hhked to she antibody polypeptide nucleic acid. Promoter sequences are known for eakaryotes. Virtually all eukaryo c genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases u stream from the start of transcription of many genes is a CNCAAT region where may ' be any nucleotide. At the 3' end of most eukar otic gett.es is an AATAAA sequence that may be the signal for addition of the poly A tai l, to the 3' end of the codin sequence.
  • Antibody polypeptide- trauseriptioa from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes ofviftises such as polyoma virus, fowipox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus, and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the aetin promoter or an j mnnmoglobuhn promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • promoters obtained from the genomes ofviftises such as polyoma virus, fowipox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sar
  • the earl and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin, of replication.
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindfH E restriction fragment.
  • a system for expressing DNA in mammalian, hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat No. 4,419,446, A modification of this system is described in U.S. Pat. No. 4,60 ! , 978.
  • the- Rous Sarcoma Virus long terminal repeat can be used as the .promoter.
  • Enhancer Element Component Traoscriptioa of DNA encoding the antibody polypeptide of this invention by higher cukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (giobin, elastase, albumin, a-fetoproteio, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Exam l s include the SV40 enhancer on the late side of fee replication origin (bp 300-270), die cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Mature 297: !
  • the enhancer may be spliced into the vector at a position 5' or 3' to the antibody porypepiide-encoding sequence, but is preferably located at a site 5' from the promoter.
  • Transcription Termination Component Expression vectors used in eukaryotic host ceils will typicall -also contain -sequences necessary for flie termination of transcription and for stabilising the inR A. ' Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DMAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding an antibody.
  • One useful transcrip tion termination component is the bovine growth hormone poiyadenyl tion region. See W094/1 1026 and the expression vector disclosed therein.
  • Suitable host ceils for cloning or expressing the DNA in the vectors herein include higher eukaryote cells described herein, including vertebrate host cells.
  • Propagation of vertebrate cells in culture has become a routine procedure.
  • useful mammalian host, cell lines are monkey kidney CVi line transformed by SV4 (COS-7, ATCC C L 1651); human embryonic kidney line (293 or 293 cells subcJoned for growt i suspension culture, Graham et aL .1, Gen. Virol 36:590.911 ⁇ : baby hamster kidney cells (BMK,. ATCC CCL 1:0); Chinese hamster ovary cells/-.DHFR (CHO, Urlaiib et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Bioi. Reprod.
  • monkey kidney cells CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma ceils (HELA, ATCC CCL 2); canine kidney cells ( DC , ATCC CCL 34); buffalo rat liver ceils (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep 02, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR.I ceils (Mather et aL Annals N.Y. Acad.
  • Host ceils are transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. [0073 ⁇ Culturing She Host Cells.
  • the host ceils used So produce an antibody of ibis invention ma be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma), Minima!
  • any of these media may be used as culture media for the host cells. Any of these media may b -su plement d as isecessary wi th hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAM GI TM drug), trace elements (defined as inorganic compounds usual ly present at final concentrations in the rmcroroolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known, to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used wish the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the antibody can be produced msraeellisiarly , or directly secreted into She medium. If the antibody is produced intraeelluiarly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by ceotrihigation. or ultrafiltration.
  • superaatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicoo or Millipore Pe!licon ultrafiltration unit
  • a protease inhibitor such as PMSF may be included is arty of the foregoing- steps to inhibit proteolysis and .antibiotics may be included to prevent the growth of adventitious contaminants.
  • the antibody composition prepared from the ceils can be purified using, for example,
  • Protein A can be used to purify antibodies that are based on human ⁇ ! , y:2, or ⁇ 4 heavy chains (Lradmaik ei aL, 1 Immunol, Meth. 62: 1-13 (.1983)). Protein G is recommended for all mouse isotypes and for human y3 (Guss ei al., EMBO J. 5: 15671575 (1986) ⁇ .
  • the -matrix to which the affinity ligand is attached is most often agarose, hut. other matrices are available. Mechanically stable matrices such as controlled pore glass or poiy(styrenedivi:t)yl)ben3 ⁇ 4ette allow for taster How rates and. shorter processing times than can be achieved with agarose.
  • the antibody comprises a CH3 domain
  • the Bafcerbond ABXTM resin (I T. Baker, Phiilipsburg ' NJ.) is useful, for purification.
  • Other techniques for protein, purification such as fractionation on. an ion-exchaage commit, ethanol precipitation, Reverse Phase HPLC, chromatography on silica,
  • some embodiments of the compounds of the invention have an affinity for human mesotheiln within identified ranges as measured in conventional assays described below.
  • Human mesothelin for use in such assays may be obtained from commercial sources, e.g. R&D Systems
  • Binding affinity means the strength of the sum total of noncovending interactions ' between a single binding site of a molecule ⁇ e.g., an antibody binding compound) ' and its binding partne (e.g. , an antigen). Unless indicated otherwise, as used herein, ' "binding affinity” refers to intrinsic binding affinity which reflects a 1: 1 interaction between members of a binding pair (e.g., antibody binding compound and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Desirably the d is 5 «10-7, !
  • Affinity can. be measured by common methods .known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer, A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative embodiments are described in the following.
  • the " d" or "Kd value" according to this invention is measured by a radiolabeled antigen binding assay (RiA) performed with the Fab version of an antibody of interest and its antigen as described by the following- assa that measures solution binding affinity of Fabs for antigen by equilibrating Fab with a minimal concentration of (i25i)-tabeled antigen in the presence of a titration series of unlabeled antigen, the capturing bound antigen with an anti-Fa b antibody-coated plat e (Chen, et al, (1999) j. Mot, Biol. 293:865-881).
  • RhA radiolabeled antigen binding assay
  • microliter plates (Dynex) are coated overnight with 5 pg nil of a capturing anti-Fab antibody (Cappei Labs) in 50 mM sodium carbonate (p ' H 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin, in FSS for two to five hours at room temperature (approximately 23° C).
  • a non-adsorbent plate (Nunc #269620) 100 pM or 26 pM [125.1]- antigeii are mixed with serial dilutions of a Fab of interest (e.g.. consistent with assessment of at) anti-VEGF antibody.
  • Fab- 12 in Presta et aL ( 1 97) Cancer Res. 57:4593-4599).
  • the Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g. , 65 hours) to insure that
  • emiilibriitm is readied. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with.0.1% Tween-20 in PBS. When the plates have dried, 150 ⁇ /well of scuniliant (MicroScint-20; Packard) is added, and the plates are counted on a Topcount gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competiti ve binding assays.
  • the Kd or Kd value is measured by using surface plasmon resonance assays using a BiAcoreTM-200 or a BiAeoreTM-30Q0 (BIAcore, Inc., Piscataway, RJ.) at 25° C. wit immobilized antigen CMS chips at " 10 response mass (RU).
  • CMS, BIAcore Inc. ⁇ carboxymet ylated dextran biosensor chips (CMS, BIAcore Inc. ⁇ are activate with -etSry!-N ' '-(3-di nesSiyia aHiopropyi)-catt!Odii
  • Antigen is dihtted with 10 niM sodium acetate, pH 4.8, into 5 pg o l ( " 0.2 ⁇ ) before injection at a Slow rate of 5 ⁇ /minate to achieve approxiioateiy 10 response anus (RU) of coupled protein.
  • IM efhanoianiine is injected to block latreaeted groups.
  • the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensit (excttaiion ⁇ 295 am; emission ⁇ 340 nm, 16 nia band-pass) at 25° C , of a 20 nM ami-antigen antibody (Fa b form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a.
  • spectrometer such as a stop-iiow equipped spectrophometer (A vi v instruments) or a 8000-series SLM-Anrinco spectrophotometer (ThermoSpectronk) with a stir red cuvette.
  • An "on-rate” or “rate of assoc iation” or “association, rate” or “kon” according to this invention can also be determioed with the same surface plasmon resonance technique described above using a BIAcoreTM- 2000 or a BIAcore 'rM -3000 (BIAcore, inc., Piscataway, M.J.) at 25 s C. with im obil&ed antigen CM5 chips at " 1 response units (RU). Briefly, carboxyrnethylaied dextran biosensor chips (CMS, BIAcore inc.
  • Antigen is diluted with 10 raM sodium acetate, pH 4.8, into 5 ug ml fQ nM) before injection at a flow rate of 5 ⁇ niinttte to achieve
  • the on-rate is preferably determined by using a fluorescent quenching technique that measures the increase o decrease in fluorescence emission intensity (e3ieitaii0n°"295 ntn; emission"340 nm, 16 ntn band-pass) at 25* C.
  • a 20 uM anti-antigen antibody Fab form
  • PBS pH 7.2
  • a spectrometer such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 800 -series SIM- Atninco spectrophototueter (TheraioSpectronie) with a stirred cuvette
  • the invention also provides tttuttunocoftjugates (interchangeably termed “antibody-dru conjugates” or "ADC”), comprising any of the antibody or antibody fragments described herein conjugated to a cytotoxic agent such as a ehemotiterapeutic agent, a drag, a growth inhibitory agent, a toxin (e.g., as enzymatieaHy active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or & radioactive isotope (i.e., a radioeonjugate).
  • a cytotoxic agent such as a ehemotiterapeutic agent, a drag, a growth inhibitory agent, a toxin (e.g., as enzymatieaHy active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or & radioactive isotope (i.e., a radioeonjugate).
  • Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as
  • cytotoxic and cytostatic effects by mechanisms including tubulin binding, D A binding, or topoisomerase Inhibition.
  • Some cytotoxic drugs tend io be inactive or less active when conjugated to large antibodies or protein receptor ligands.
  • ZEVALIN® ibritttmomab tiuxetan, Biogen/idec
  • ZEVALIN® is an antihody-fadioisotope conjugate composed of a murine lgGI kappa monoclonal antibody directed against the CD2 antigen found on die surface of normal arid malignant B lymphocytes and 11 Hn or 0Y radioisotope bound by a thiourea Imker-chelator (Wiseman et al., (2000) Eur. Jour. ttci. Med. 27(7 ⁇ :?66-77; Wiseman et al., (2002) Blood 99(12);4336-42; Witag et at, (2002) J. Clio.
  • ZEVALIN has activity against B-ceil non-Hodgkin's Lymphoma (NHL), administration .results in severe and prolonged cytopemas in. most patients.
  • MYLOTARGTM gemtuzurnab ozoganticsu, Wyeth Pharmaceurkals
  • an antibody drug conjugate composed of a hn CD33 antibody linked to eaiichearaichi was approved in 2000 for the treatment, ofacute myeloid leukemia by injection (Drugs of the Future (2000) 25(7):6 6; U.S. Pat.
  • Caotimtmab raertaftsine (In nogen, lac. ⁇ , an antibody drug .conjugate composed of the huC242 antibody linked via the disulfide linker SPP to the roaytansinoid drag moiety, DM 1 , is approved for the treatment of cancers that express CanAg, such as colon, pancreatic, gastric, and others.
  • MLN-2704 an antibody drug .conjugate composed of the huC242 antibody linked via the disulfide linker SPP to the roaytansinoid drag moiety, DM 1 .
  • PSMA monoclonal antibody linked to the maytansinoid drug moiety, DM i, is under development for the potential treatment of prostate tumors.
  • auristatin peptides auristatin E (AE) and monomethyiauristafin (MMAE), synthetic analogs of doiastatin, were conjugated to chimeric monoclonal antibodies cB .96 (specific to Lewis Y on carcinomas) and cACIO (specific to C.D30 on hematological malignancies) (Doronina et al., (2003) Nature Biotechnology 2! (7): 778-784) and are under therapeutic development.
  • AE auristatin E
  • MMAE monomethyiauristafin
  • Cbemotherapeudc agents useful in die generation of inimnuoconjagates are described herein (e.g., above) .
  • En ⁇ matiealiy acti ve toxins and fragments thereof that can be used include diphtheria A chain, nonblnding active fragments of diphtheria toxin, exotoxin.
  • a chain (from.
  • Pseudoraonas aeruginosa ricin A chain, abrin A chain, modeechi A chain, aipha-sarchi, Al.eurites fordii proteins, dianthin proteins, Phyiolaca americana proteins (FAPI, PA.Pli, and PAP-S ⁇ , momordica charantia inhibitor, curcia, crotia, sapaoaaria officinalis inhibitor, gelonin, ⁇ mitogei.iin, restrieiociu, phermniyein, enoniycin, and the trieofhecenes. See, e.g., WO 93/21232 published Oct. 28, 1993.
  • radionuclides are available for the production of radioconjugated antibodies. Examples include 2 ! 2B . 1311, 131 ⁇ , 90Y. and 186S.e, Conjugates of the antibody and. cytotoxic agent are made using a variety ofbiftinctional protein-coupling agents such, as - succiimnklyl-3- ⁇ 2-pyrid 1dithiol) propionate (SPDP), tmmotbiolane (IT), bifurictional derivatives of iinidoesters (such as dimethyl, adipinridate HCl), active esters (such as disuceininiidyl suberate), aideliyde (suc as glntanddetryde), bis-azido compounds (such as bisip-aadobenzoyl) hexanediamtne), bis-dtazomum derivatives (such as bis ⁇ pp ⁇ liammmnbetwyi)-e
  • a .ricin. i munotoxin can be prepared as described in Vitet a et al., Science,. 238; 1098 ( 1987).
  • Carbon-14- iabeted 1 -iKothiocyaaatobeazykV e ⁇ triaminepentsacetie acid (MX-DTPA) is aa exem lars' chelating agent for conjugation ofradionucleotide to the antibody. See 094/1 1026,
  • Conjugates of an antibody and one or more snail molecule toxins such as a ealicbeanncin, maytansinoids- dolastaiins- aurostatins, a trichothecene, and CC106S, and the deri vatives of these toxins that have toxin activity, are also contemplated berein.
  • ADC ttie antibody binding conijsoand- drug conjugates
  • an antibody is conjugated to one or more drag moieties (D), e.g. about 1 to about 20 drug moieties per antibody binding compound, through a linker (L).
  • D drag moieties
  • L linker
  • the ADC: of Formula I may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophitie group of an antibody with a bivalent linker reagent to form Ab-L, via a covaiera bond, followed by reaction with a drug moiety D; and (2) reaction of a nitc ieophiiie group of a drug moiety with a bivalent linker reagent, to form D-L, via a covalent bond, followed, by reaction with the nucleophilic group of an antibody.
  • the linker may be composed of one or more linker components.
  • exemplary linker components include 6-maieimidoeaproy.t ("MC"), maleiraidopropanoyi (“MP”), va.line-citndl.ine Cvai-eit”), alanine- phenylalanine ("ala-phe"), p-aminobeiizyloxycarbonyl ("PAB " ), N-Succimniidyl 4-(2-pyridylthio)pentanoate C'SPP”), N- ueeinimidyl 4-(N-malein)idomeihyl)cyciohex3ne ⁇ i earboxyiaie (“SM.CC”), and N- ueeinimidyi ⁇ 4-iod.o-acetyi)ai:n.iiiobeniioate C'SIA.B”).
  • MC 6-maieimidoeaproy
  • the linker may comprise amino acid residues.
  • exemplary amino acid linker components include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
  • Exemplary dipeptides include: valine-citridline (vc or val-cit), alarrine-phenylalamne (afor ala-phe).
  • Exemplary tripeptides include: glycine- v line-ciirulline (giy-vai-cit) and glycine-glycine-glycine-glycine (giy-giy-gly)-
  • Amino acid residues which comprise an amino acid linker component include those occurring naturally, as well as minor amino acids and non- naturally occurrin ammo acid analogs, such as ci!ruiiine.
  • Amino acid I ker components can be designed and optimized in. their selectivity for enzymatic cleavage by a particular enzymes, for example, a tumor- associated protease, cathepsin B, C and D, or a plasmin protease.
  • Nucleophiiic groups on antibodies include, but are not limited to: (i) N-terrainai amine groups, (si) side chain amine groups, e.g. .lysine, (iii) side chain thiol gamps, e.g. cysteine, and (iv) sugar hydroxy! or amino groups where the antibody is glycosylated.
  • Amine, thiol, and hydroxy! groups are nuc.ieop.hiHc and capable of reacting to form covending bonds with electrophiiic groups on linker moieties and linker reagents including: fi) active esters such as NHS esters, HOBt.
  • esters, haloformates, and acid haiides (ii) alkyl and benzyl haiides such as haioacetatiiides; (iii) aldehydes, ketones, carboxyl, and maieimide groups.
  • Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges.
  • Anti odies may be made reactive tor conjugation with linker reagents by treatment with a reducing agent such as DTT (dinhofhreitol), Each cysteine bridge will thus form, theoretically, two reactive thiol nacleophiles.
  • Additional nucleophiiic groups can be introduced into antibodies through the reaction of lysines with 2-i mothiolane (Traut's reagent) resulting in con version of an amine into a thioi.
  • Reactive thioi groups may be introduced into the antibody (or fragment thereof) b introducing one, two, three, four, or more cysteine residues (e.g., preparin mutant antibodies comprising one or more non-native cysteine amino acid residues).
  • Antibod drug conjugates of the invention may also be produced by modification of the antibody to introduce electrophiiic moieties, which can react with nucleophiiic substitueats on the linker reagent or drug.
  • the sugars of glycosylated antibodies may be oxidized, e.g., with periodate oxidizing reagents, to form aldehyde or ketone groups which may reac with the amine group of linker reagents or drug moieties.
  • the resulting inline Sebiff base groups may form a stable linkage, or may be reduced, e.g., by borobydride reagents to form stable amine linkages.
  • reaction of the carbohydrate portion of a glycosylated antibody with either glactose oxidase or sodium meta-periodaie may yield carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug (Hermanson,
  • proteins containing N-tenninal serine or threonine residues can react wi th sodium meta-periodate, resulting in production of an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852).
  • aldehyde can he reacted with a drug moiety or linker iiacleophile.
  • nucleophiiic groups on a drug moiety include, bat are not limited to: amine, thiol, hydroxy!, hydrazide, oxime, hydrazine, thioseaiiearbazone, hydrazine carboxylase, and aryl!iydrazide groups capable of reacting to form covending bonds with electrophiiic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters.
  • HOBt esters haloibrraates, and acid haiides
  • alkyl and benzyl haiides such as haloaeetamSdes
  • aldehydes ketones, carboxyl, and maleimide groups.
  • a fusion protein comprising the antibody and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis.
  • the length of DMA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.
  • ie antibody may be conjugated to a "receptor" (such strepta.vidin) for utilization in tumor pre-targeting wherein the antibody-receptor conjugate is administered to the individual, followed by removal of unbound conjugate frorn tiie circulation using a clearing agent and then
  • a "hgand” e.g., avidin
  • a cytotoxic agent e.g., a radioffiseieotide
  • the antibody binding regions may be fused with T ceil transmembrane and signaling components to form a chimeric antigen receptor (CAR), as described more fully above.
  • CAR chimeric antigen receptor
  • Therapeutic formulations comprising an. antibody binding compound of the invention are prepared for storage by mixing Site antibody having Site desired degree of purity with optional physiologically acceptable earners, excipients or stabi lizers (Remington: The Science arid Practice of Pharmacy 20th edition (2000)), in the form of aqueous solutions, iyophilized or other dried formulations.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffets such as phosphate, citrate, histidiae and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as ociadeeyidiroethylberayl ammonium chloride; hexamethoniitm chloride; benzalkonmm chloride, benxethonium chloride; phenol, butyl or benzyl alcohol; alkyl parahens such as methyl or propyl paraben; catechol; resorchioi: cyclohexanol; 3-peiitanol; and m-eresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or
  • miiiiunoglobulins hydrophilic polymers such as polyvinylpyrrolidone; annuo acids such as glycine, giatamine, asparagine, hisridinc, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, raannose, or dextrius; chelating agents such as EDTA; sugars such as sucrose, taaanitoi, trehalose or sorbitol; salt-forming counter-ions such as sodi m; metal complexes (e.g., Zn-protein complexes); and or non-ionic surfactants such as TWEENTM, PLURONlCS rM or polyethylene glycol (PEG),
  • the fbnrmlation herein may also contain more than one active compound as necessary for the particular 'indication being treated, preferably those with coumtementary activities that do not adversely affect each other.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by inietfacial polymerization, for example, hydroxyniethylcellulose or gel tia-inicrocapsule and poly-(oiethylinethacykue) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, raicroetanlsions, nano-particles and nanocapsules) or in rxiacroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, raicroetanlsions, nano-particles and nanocapsules
  • rxiacroemulsions for example, liposomes, albumin microspheres, raicroetanlsions, nano-particles and nanocapsules
  • Such techniques are disclosed in Remington; The Science and Practice of Pharmacy 20th edition (2000), [ 098 ⁇
  • sustained-release preparations may be prepared.
  • suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the immunoglobulin of the invention, which matrices are in. the .form of shaped articles, e.g., films, or microcapsule.
  • sustained-release matrices include polyesters, hydrogeis (for example, poly(2-hydro ⁇ emyl-methacn'late), or poiy(vinyiaicohol)), polylactid.es (U.S. Pat. No.
  • antibody binding compounds of the invention are administere to a human patient, in accord with own methods, such as intravenous administration as a bolus or by continuous infusion over a period, of time, by intramuscular, intraperitoneal, intracerohrospiuaL
  • subcutaneous, intra-art cniar, intrasynovial, intrathecal, oral, topical, or inhalation routes, intravenous or subcutaneous administration, of the antibody is preferred.
  • Method of treatment using compounds of the present invention involves the administration of an antibody binding compound of the invention to an animal or human patient, followed at. intervals by subsequent doses of equal or smaller doses such that a target serum concentration is achieved and maintained during treatment.
  • maintenance doses are -delivered by bolus -delivery, preferably b subcutaneous bolus administration, makin treaonent convenient and cost-eSTeetive for the patient and health care professionals,
  • chemotherapeutic agent other than an aniracycl e
  • the combined administration includes co-administration, using separate iormulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities.
  • Preparation and dosing schedules for such ehemotherapeutic agents ma be used according to maraifacturers' instructions or as determined, empirically by the skilled practitioner. Preparation and dosing schedules .for such
  • the eheruotherapeutic agent may precede, or follow administration of the antibody binding compound or may be given simultaneously therewith.
  • I Mg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of antibody is an initial candidate dosage for administration, to the patient, whether, for example, by one or more separate administrations, of by continuous inmsion.
  • a typical daily dosage might range from about ⁇ ,ttg/kg to 100 rag/k or more, depending on the factors mentioned above.
  • dosage regimens may include an initial dose of antibody binding compound of 6 mg/kg, 8 mg/kg, or 12 mg/kg delivered by intravenous or subcutaneous infusion, followed by subsequent weekly maintenance doses of 2 mg/kg by intravenous infusion, intravenous bo!us injection, subcutaneous infusion, or subcutaneous bolus injection. Where ' the antibody is well-tolerated by the patient, the time of infusion may be reduced,
  • the invention includes an initial dose of 12 mg/kg antibody binding compound, followed by subsequent m intenance doses of 6 mg kg once per 3 weeks,
  • Another dosage regimen involves an initial dose of 8 rag/kg antibody binding compound, followed by 6 mg/kg once per 3 weeks.
  • Still another dosage- egimen involves an initial dose of 8 mg/kg antibody binding compound, followed by subsequent maintenance doses of 8 tag/kg once per week or 8 mg/kg once every 2 to 3 weeks,
  • initial doses of 4 mg/kg antibody binding compound may be administered on each of days 1, 2 and 3, followed by subsequent mainteaaace doses of 6 mg/kg once per 3 weeks.
  • An additional regimen involves an initial dose of 4 mg/kg antibody binding compound, followed by subsequent maintenance doses of 2 mg/kg tw ice per week, wherein the maintenance closes are separated by 3 days.
  • the invention may include a cycle of dosing in which delivery of antibody binding compound is 2-3 times per week for weeks.
  • the 3 week cycle is preferably repeated as necessary to achieve suppression of disease symptoms.
  • the invention farther includes a cyclic dosage regimen in which delivery of antibody binding compound is daily for 5 days. According to the invention, the cycle is preferably repeated as necessary to achieve suppression of disease symptoms.
  • This combinatorial phage library was then subjected to three rounds of selection using progressively stringent wash times (4hours, 18 hours and 48 hours ⁇ . The resultant phage were then cloned and individual clones were sequenced. Several specific mutations were represented in a majority of the recovered clones and six clones were chosen for further study. Each clone was grown up as phage, bound to mesothe-Sin and .subjected to a short, 5 ⁇ wash and a long 48 hour wash.
  • the phage were recovered from, each of these binding reactioas using an acid elution and the number of bound phage in each sample was quantised via qPCR , Each of the six selected variants showed a much greater retention it) the stringent 48 hour wash than the S D I parent, compound.
  • Samples of the six clones were then heated to 65 3 ⁇ 4 for one hour and then thei binding to niesothehn was tested against a parallel sampl of the unhealed phage. Again the mimber of bound phage in each of the binding reactions was measured via qPCR.
  • the parental SDI phage and one of the variants, D8, showed good binding following the 65°C heat treatment.
  • the other 5 variants showed a significant loss of bindin activity (30-300 fold). Given the increased affinity and good thermal stability, D8 was selected as a promising therapeutic candidate for the treatment of cancers with mesotheiin overexpression.
  • the number of rescued phage from each sample ' was determined using a SyberGreen qPCR amplification reaction with, fee primers CmF2 (5" TTTCCGGCAGTTTCTACAC .V) and CmR.1 (5' CAGCACCTTGTCGCCTTGC V) on a Applied Biosystems StepOnePius Re&i-iime PCR. system using a standard curve with phage diluted in PBS at 3s f, 3 x ⁇ 3 10 6 , 3 X 10 s , 3 x L0 '! and 0 phage/well. Data is shown in Fig, IB.
  • 100117 ⁇ protein either natural, or pa heticall ' ' produced by recombinant or chemical tneans (but the design of whose antigen bindi ng region, is derived from a natural counterpart), which is capable of specifically binding to particular antigen or antigenic determinant
  • the binding characteristics, e.g. specificity, affinity, and the like, of an antibody, or a binding compound derived from an antibody are determined by amino acid residues in me VH and/or V . regions, and especially in the three to six complementary determining regions (CDRs).
  • the constant domains are not involved directly is binding an antibody to art antigen.
  • immunoglobulins can be assigned to different classes.
  • imm itogiobahns There are Five major classes of imm itogiobahns: igA, I D, igE, fgO, and IgM, and several of these can be further divided into subclasses (tsotypes). e.g., IgG 5 , igG 3 ⁇ 4 . IgOj, IgG. ⁇ , IgAs, and igA?.
  • ADCC antibody-dependeni cell-mediated- cytotoxicity'
  • FeRs present 0(1 certain cytotoxic ceils (e.g.. Natural Killer (MK) cells, neutrophils, and macrophages) enable these cytotoxic effector ceils to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxim.
  • MK Natural Killer
  • the antibodies “arrt * the cytotoxic cells and are absolutely required for such killing.
  • f>,737, 56 may be- erformed.
  • Useful effector eeils for such assays include peripheral blood mononuclear cells (PB C) and Natural Killer (N ) ceils, Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in ;i animal model -such as that disclosed ' in Clynes et C PNAS (LISA) 95:652-656 (1998).
  • Antibody fragment as used herein are defined as a portion of an intact antibody comprising the antigen binding site or variable region of the intact: antibody , wherein tne portion is free of the constant heavy chain doroains (i.e. CB2, CB3, and Q-I4, depending 011 antibody isotype) of the Fc region of the intact antibody.
  • constant heavy chain doroains i.e. CB2, CB3, and Q-I4, depending 011 antibody isotype
  • antibody fragments include Fab, Fab', Fab'-SH, F(ab')3 ⁇ 4 and Fv fragments; diabodles; airy antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment” or “single chain polypeptide”), including without limitation (!) single-chain Pv (scFv) molecules (2) single chain polypeptides containin only one light cha n variable d ma n, or a 'fragment thereof that contains .the three CDRs of the light chain variable domain, without art associated heavy chain moiety and ⁇ single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated Sight chai moiety; and maihspeci c or nmltivalent structures formed from antibody fragments.
  • Binding affinity generall refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and it binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity” refers to intrinsic binding affinity which reflects 1:1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd), Desirably the Kd is i * 10-7, 1 * 10-8, 5*10-8, I * 10-9, 3* 10-9, 5* 10-9, or even ! * 10-10 or stronger.
  • Affinity can be measured by common methods known in. the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tead to dissociate readily, whereas high-affinity antibodies generally bind antigen fester and lend to remain bound longer.
  • a variety of methods of measuring binding affinity arc known ia the art any of which can be us ed for purpos es of tbe present invention. Specific illustrative embodiments are described ia the following.
  • Binding compound means a compound that is capable of specifically binding to a particular target molecule or group of target molecules.
  • Antibody binding compound m ns a binding compound derived from an antibody, such as an antibody fragment, including but not limited to, Fab, Fab ' , F(ab1 ⁇ 4 and Fv fragments, or recombinant forms thereof, in some embodiments, an antibody binding compound may comprise a scaffold or framework regions of one antibody nd one or more CDR regions of another antibody.
  • Chimeric antigen receptor refers to a recombinant bioroolecuie that contains an extracellular recognition domain, a transmembrane region, and an intracellular signaling domain.
  • the extracellular recognition domain also referred to as the extracellular domain or simply by the recogni tion element which it contains) comprises a recognition, element that, -speciiicaify binds to a molecule present on the cell surface of a target cell, which comprise an antibody binding compound, of the invention.
  • the transmembrane region anchors tbe CAR in. the membrane.
  • the intracellular signaling domain may comprise a signalin domain, such as (but not limited to) a signaling domain from the a chain of the human CDS complex, aad optionally " Comprises one or more co-stumilatory signaling domains.
  • CompleniCBtary-deierraining region or "CDR” means a short Sequence (from 5 to IS amino acids) in (he variable domains of immunoglobulins. These regions are also referred to herein as
  • the CDRs (six of which are presen t ia IgG molecules) are the most variable pan of immunoglobulins aad contribute to their diversity by making sped fie contacts with a specific antigen, allowing itmnunogiobaims to recognize a vast repertoire of antigens with a high affinity, e.g. Beck et al, Mature Reviews immunology, 10: 345-352 (2010), Several numberin schemes, such as the Kabat
  • CDRs Complementarity Determimng Regions
  • the Kabat Complementarity Determimng Regions are based on sequence variability and are tbe most commonly used (Kabat et al., Sequences of Proteins of immunological Interest, 5th Ed. Public Health Service, National institutes of Health, Bethesda, Md. (1991)). Chothia designations refer to the location of the structural loops (Chothia and Lesk. I Mel, Biol. 196:901 -917 (1987)).
  • CDR M as used herein may include the Kabat definition, or the Chothia definition of amino acid locations of such regions, of me term may include other definitions as described or expressly set forth herein, in some embodiments, ⁇ .lie invention, includes CDRs that include amino acids beyond those defined in she Kabat or Chothia naming conventions,
  • Cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of ceils.
  • the term is intended to include radioactive isotopes (e.g., Ai2i 1 , 1135 , IS 25, Y'30, Re186, Re 188, Sml 53, Bi 12, P32, and radioactive isotopes of Ly), ehemotherapeuiic agents e.g., methotrexate, adriatnicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunofubiciii or other iasercalatirig agents, enzymes and fragments thereof such as mtcleolyhc enzymes, antibiotics, and toxins such -as small molecule toxins or emyroatkally aciive toxins of bacterial
  • radioactive isotopes e
  • Eifector functions of antibodies refer to those biological activities attributable to the Fc region (a native sequence Fe region or amino acid sequence variant Fc region.) of an anti body, and vary with the antibody isotype.
  • antibody effector functions include; Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of ceil surface receptors (e.g., B cell receptor); and B ceil activation.
  • Fab fragment contains the constant domain of the Sight chain and die first constant domain (CHI) of the heavy chain
  • Fab ! fragments differ from Fab fragments by die addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region
  • Fab'-SH is the designation herein for f b' in which the cysteine residue(s) of die constant domains bear a free thiol group.
  • F(ab' ⁇ 2 anti body fragments originally were produced as pairs of Fab' .fragments which have hinge cysteines between them.
  • TR n residues are those variable domain residues other titan the CDR or hypervariabie region residues as herein defined.
  • a "human consensus framework” is a framework which represents the most commonly occurring amino acid residue in a selection of human immunoglobulin V.L or VH framework sequences.
  • Fv fragment is the .minimum antibody fragment which contains a. complete antigen-recognition and -binding site, in a. two-chain Fv species, this region consists of a dimer of one heavy- and one light-chain variable domain, in tight, non-eovalent association, in. a single-chain Fv species, one heavy- and one light' chain variable domain can be covalentiy linked by a flexible peptide linker such that the light and heavy chains can associate in a "dinierie" structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain, interact, to define an. antigen-binding site on the surface of the VH-VL dimer.
  • the six CDRs confer antigen-binding specificity to the antibody.
  • “Humanized” forms ef non-human (e.g., murine) antibodies are chimeric antibodies that contain, minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human in'iMunoglobulms ⁇ recipient antibody) in which, residues from a hypervariabie region of the recipient are replaced by residues from a hypervariabie region o f a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • framework region (F.R) residues of the human immunoglobulin are .replaced by corresponding nonhuman residues.
  • humanized antibodies may comprise residues that are not found is the recipient antibody or in th - donor antibody. These modifications are Bade to further refine antibody periormaace, la general, the humanized antibody will comprise substa-ttialry aii. of at Ieasi one, and typically two, variable domains, m which all or substantially all of the hypemsria ie loops- correspond to those of a non-human Itfliouxtogiooutiii and -all f substantially ail of the FRs are shose of a .human hnoiunogtobu m sequence.
  • the humanized antibody optionally will also comprise at least a.
  • n immunoglobulin constant region typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • an antibody or antibody binding compound in. reference to an antibody or antibody binding compound means such a compound which, has been identified and separated and/or recovered from, a component of its natural environment, or from a heterogeneous reaction mixture. Contaminant componeais of a natural environment or reaction mixture are materials which would interfere with diagnostic or therapeutic uses for the antibody or antibody binding compound, and may Include enzymes, hormones, and other proteinaceous or nonpreteinaceous solutes; to some embodiments, an antibody or antibody blading compound is ' purified ( j) to greater man 95% by weight of antibody or antibody binding compound as -determined by the Lo ry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain, at least 15 residues of N-terminai or internal amino acid sequence by use of a spinning cup sequenafor, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • an isolated antibody or antibody binding compound may include the antibody or antibody binding compound in. situ within recombinant cells since at least one component of the antibody's or antibody binding compound's natural environment will not be present. Ordinarily, an isolated antibody or isolated antibody binding compound is prepared by at least one purification step,
  • mAb monoclonal antibody
  • mAb monoclonal antibody
  • mAb refers to an antibody obtained from a population of substantially homogeneous antibodies., i.e., the individual antibodies comprising the population are identieai except for possible naturally occurring mutations that may be present in minor amounts.
  • Monoclonal anti bodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture or fay bacterial, yeast or mammalian expression systems, iineont animated by other immunoglobulins.
  • Percent (%) amino acid sequence identity with, respect io a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that ate within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megaiign (DNASTAR) software.
  • ALIGN-2 sequence comparison computer program
  • Table A sequence comparison computer program
  • the ALIGN-2 Sequence comparison computer program was authored by Geaersieeh, inc.. and the source code has been filed with, user documentation in. the U.S.. Copyright Office, Washington B.C., 20559, where it is registered under U.S.
  • the ALIGN-2 program is publicly available through Genentech, inc., South San Francisco, Calif, or may be compiled from the source code provided in, e.g., WO200? (K)iS51.
  • the ALIGN-2 progra should be compiled for use on a UNIX operating system, preferably digital UNIX V4.GD, All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is employed for ammo acid sequence comparisons, the % amino -acid sequence identity of a given amino acid sequence A to. with, or against a given amino acid sequence B (which can alternatively be phrased as a giveo amino.
  • acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction ⁇ , where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in tot program's alignment of A and B, and where Y is the total number of amino acid residues in. B, It will lie appreciated tha t where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
  • two or more amino acid sequences are at least 50%, 60%, 70%, 80%, or 90% identical. More desirably, two or more amino acid sequences are at least 95%, 97%, ⁇ 8%, 99%, or even 1 0% identical. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program,
  • (Mil 33 j "Phage display' * is a technique by which variant polypeptides are displayed as fusion proteins to at least a portion of a coat protein, on the surface of phage, e.g.,.. filamentous phage, particles
  • a utili ty of phage display lies in the fact that large libraries of randomized protein variants can be rapidly and efficiently selected for those sequences that, bind to a target molecule with, high affinity. .Display of peptide and protein libraries on phage lias been used for screening millions of polypeptides for ones with specific binding properties.
  • Polyvalent phage display methods have been used for displaying small random peptides and small proteins through fusions to either gene III or gene VIII of filamentous phage.
  • a protein . peptide library is fused to a gene Hi or a portion thereof, and expressed at lo w levels in the presence of wild type gene Hi protein so thai phage particles display one copy or none of the fusion proteins.
  • Phagemicr means a piasmid vector having. a bacterial origin of replication, e,g Berry Col El , and a copy of an allergenic region of a bacteriophage.
  • the pfaagermd may be used on. any known bacteriophage, including filamentous bacteriophage and lanibdoid bacteriophage.
  • the piasmid will also generally contain a selectable marker for antibiotic resistance. Segments of DNA cloned into these vec tors can be propagated as pksmids.
  • Hie phage mid may form infectious or noninfectious phage particles.
  • This terra includes phageraids, which contain a phage coat protein gens or fragment (hereof linked to a heterologous polypeptide gene as a gene fusion such (hat the heterologous polypeptide is displayed on the surface of the phage part icle.
  • phage vector means a doable stranded, epltcaiive form of a bacteriophage containing a heterologous gene and capable of replication.
  • the phage vector has a phage origin of replication allowing phage replication and phage particle formation.
  • the phage is preferably a filamentous
  • bacteriophage such as an M ⁇ 3, f 1 , fd, PB phage or a derivative thereof, or a lambdoid phage, such as lambda, 21, pbiSO, phi8l, 82, 424, 434, etc., or a derivative thereof:
  • Polypeptide refers to a class of compounds composed of amino acid residues chemically bonded together by amide linkages with elimination of water between the carboxy grou of one amino acid and the amino group of another amino acid.
  • a polypeptide is a polymer of amino acid residues, which may ' contain a large number of such residues.
  • Peptides axe similar to polypeptides, except that, generally, they are comprised of a. lesser number of amino acids. Peptides are sometimes referred to as oligopeptides. There is no clear-cut distinctio between polypeptides and peptides. For convenience, i this disclosure and claims, the term "polypeptide* * will be used to refer generally to peptides and polypeptides.
  • the amino acid residues may be natural or synthetic.
  • the scFv polypeptide further comprises a polypeptide linker between the VH and. VL domains which enables the scFv to form the desired structure for antigen binding.
  • molecules involved in a specific biading event have areas oa their surfaces or is cavities giving rise to specific recognition between the molecules binding to each other.
  • specific binding include antibody-antigen interactions, enzyme-substrate interactions, formation, of duplexes or triplexes among polynucleotides and/or oligonucleotides, receptor-ligand interactions, and the like.
  • contact in reference to specificity or specific binding means two molecules are close enough that weak noncovalent chemical interactions, such as Van der Waal forces, hydrogen bonding, base-stacking interactions, ionic and hydrophobic interactions, aad the like, dominate the interaction of the molecules,
  • Therapeutically effective amount means art amount having antiproliferative effect oa a cancer, in some embodiments, a therapeutically effective amount is an amount that induces apoptotic activity, or is capable ofmduciag ceil death, particularly death of benign or malignant tumor ceils. Such efficacy can be measured in conventional ways, depending on the condition to be treated. For cancer therapy, such efficacy can, for example, be measured by assessing the time to disease progression, or determining the response rates. Therapeutically effective amount also refers to a target serum concentration of an antibody binding compound of the invention, such as a trough serum concentration , that lias been shown to be effective in suppressing disease symptoms when maintained ior a period of time.
  • variable region or 'Variable domain' refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies, it is concentrated in three segments called complementarify-detemuning regions (CDRs) or hypervariabie regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FK.
  • a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabai j after residue 52 of HI and inserted residues (eg, residues 82a, 82b, and &2e, etc according to Kabai) after hea y chain FR residue 82.
  • the Kabai 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" Kabai numbered sequence. It is not intended that CD s of the invention necessarily correspond to the abat numbering convention.
  • Vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • ector is a "piasmid,” which refers to a c ircular double stranded DMA loop into which additional DMA segments may be Hgated.
  • phage vector Another type of vector is a viral vector, wherein additional DNA ' segments may be hgated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host ceil into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episontal mammalian vectors).
  • vectors e.g., non-episomal mammalian- vectors
  • vectors can be integrated i to the genome of a host cell upon introduction into the host, cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "recombinant expression vectors 5* (or simply, "recombinant vectors"), in general, expression vectors of utility iu recombinant DMA techniques are often iu the form of plasmids.

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Abstract

La présente invention concerne de nouveaux composés de liaison à l'anticorps SD1 et des méthodes d'utilisation de ceux-ci. Les composés de liaison à l'anticorps SD1 de l'invention comprennent de nouveaux polypeptides d'immunoglobuline à chaîne lourde ou des parties de ceux-ci.
PCT/US2016/017888 2015-02-17 2016-02-12 Nouveaux composés de liaison à sd1 et leurs utilisations WO2016133829A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050244901A1 (en) * 2002-04-22 2005-11-03 Dieter Peschen Antibodies, recombinant antibodies, recombinant antibody fragments and fusions mediated plant disease resistance against fungi
WO2014052064A1 (fr) * 2012-09-27 2014-04-03 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anticorps dirigés contre la mésothéline et procédés d'induction d'une activité anticancéreuse puissante

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050244901A1 (en) * 2002-04-22 2005-11-03 Dieter Peschen Antibodies, recombinant antibodies, recombinant antibody fragments and fusions mediated plant disease resistance against fungi
WO2014052064A1 (fr) * 2012-09-27 2014-04-03 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anticorps dirigés contre la mésothéline et procédés d'induction d'une activité anticancéreuse puissante

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