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WO1991013166A1 - Constructions d'anticorps ayant une affinite de liaison amelioree - Google Patents

Constructions d'anticorps ayant une affinite de liaison amelioree Download PDF

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Publication number
WO1991013166A1
WO1991013166A1 PCT/US1991/000633 US9100633W WO9113166A1 WO 1991013166 A1 WO1991013166 A1 WO 1991013166A1 US 9100633 W US9100633 W US 9100633W WO 9113166 A1 WO9113166 A1 WO 9113166A1
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Prior art keywords
construct
antibody
domain
binding
region
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PCT/US1991/000633
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English (en)
Inventor
Stephen D. Gillies
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Abbott Biotech, Inc.
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Publication date
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Priority to AU73334/91A priority Critical patent/AU644413B2/en
Priority to JP3505551A priority patent/JPH07507440A/ja
Publication of WO1991013166A1 publication Critical patent/WO1991013166A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • This invention relates to cancer imaging agents and cancer therapeutic agents, and more specifically, to the preparation of antibody constructs useful as imaging agents and/or cancer therapeutic agents.
  • this invention relates to biosynthetic antibody constructs with enhanced antigen binding affinities and a short half life in vivo.
  • Systemic pharmacotherapy is a commonly used mode of therapy.
  • labelling agents such as radioisotopes for tumor or organ imaging.
  • the administration of such drugs and radiolabels by themselves, is risky in that the drugs usually are not target selective.
  • Limiting the effects of chemotherapeutic drugs and radioisotopes used in the treatment of cancer are particularly difficult because these chemicals have the ability to interfere with the metabolic processes of most cells, especially those that are in a proliferative state. Therefore, the art frequently suggests coupling such drugs to an agent capable of targeting a particular tissue or cancerous cell type.
  • Antibody molecules have been conjugated to various drugs, enzymes, toxins, and radioisotopes (reviewed in Ghose and Blair (1978) J. Natl. Cancer Inst. ⁇ l:657-676) .
  • antibodies have been successfully employed to image occult tumors not detected by traditional detection methods such as magnetic resonance imaging, x-ray analysis, and computed tomography (see Goldenberg (1988) Arch. Pathol. Lab. Med. 112:580-587).
  • Radiolabeled antibodies having a specificity for tumor antigen may show accretion rates of only 0.01% to 0.001% in the targeted tumor (Goldenberg, ibid.) .
  • there may be a high level of nonspecific binding of the intact antibody despite its specificity for a particular tissue-associated antigen. Some of this nonspecific binding has been attributed to the adherence of the antibody via its Fc portion, rather than via its binding domain.
  • intact antibodies have a slow clearance rate relative to smaller molecules such as fragments of antibody molecules.
  • the level of circulating and nonspecifically bound radioactivity is high, and clearance occurs gradually over a period of four days or more (Goldenberg, ibid.) .
  • the use of antibody fragments without Fc domains permits tumor imaging within 24-48 hours, thereby permitting the use of radioisotopes with short half lives, such as 123 i and 99m Tc (Goldenberg, ibid.).
  • F(ab')2 fragments are not without pitfalls. Besides being difficult to produce enzymatically, the binding properties of these fragments may be compromised (see, e.g.. Wahl et al., ibid. Therefore, what is needed is a targeting molecule having specificity, enhanced binding activity, minimal nonspecific binding abilities, and a shorter half-life in vivo.
  • an object of the present invention to provide antibodies which recognize tumor antigens, and would therefore be useful for the imaging and/or killing of tumors. It is also an object of the present invention to provide antibody constructs useful for tumor imaging and/or cancer therapy which have reduced Fc receptor binding so that their non-specific accumulation in the spleen and other parts of the body is reduced. Additional objects include the provision of antibody constructs which can bind quickly to tumor antigens and the provision of antibody constructs with shorter half lives than intact, whole antibody molecules.
  • Native antibody molecules of the IgG and IgD classes are composed of two heavy (H) and two light (L) chains which are held together by covalent (disulfide) bonds and non-covalent interactions.
  • the variable (V) domains at the amino termini of the two chains together form the antigen binding region.
  • the first constant (C) domain of the H chain (CHI) interacts with the C region of the L chain through hydrophobic interactions and also through a disulfide bond.
  • the next H-chain domain, adjacent to the hinge is called CH2.
  • This domain reportedly contains many of the effector functions of the antibody including the sequences responsible for complement fixation and Fc receptor-mediated antibody-dependent cellular cytotoxicity (ADCC) , and is the sole N-linked glycosylation site in human C ⁇ l chains.
  • ADCC antibody-dependent cellular cytotoxicity
  • the CH3 domain at the carboxy terminus of the H-chain is thought to play a major role in the assembly of H chains.
  • the antibody constructs of the present invention include a first portion having an immunoglobulin binding region of predetermined specificity. This binding region has plural hypervariable regions homologous with hypervariable regions of a native antibody specific for the same epitope.
  • the antibody constructs further include a second portion consisting essentially of a CHI domain and a hinge whose C terminus is linked to the N terminus of a CH3 domain, i.e., a hybrid constant domain which omits the CH2 domain normally present in a native antibody.
  • Antibody constructs with the CH2 domain deleted are referred to hereinafter as ⁇ CH2 constructs.
  • ⁇ CH2 constructs are characterized by enhanced binding activity, at least twofold greater than the binding activity of an antibody construct with the same specificity but having an intact constant domain.
  • the binding region of the ⁇ CH2 antibody construct includes the binding region of a nonhuman immunoglobulin such as a murine immunoglobulin.
  • the immunoglobulin binding region is one which has a predetermined specificity for mucin such as the binding region of monoclonal antibody B72.3, or has a predetermined specificity for the G * 3 2 ganglioside such as the binding region of monoclonal antibody 14.18.
  • the immunoglobulin binding region has a predetermined specificity for a melanoma-specific proteoglycan.
  • the preferred altered constant region includes a human immunoglobulin constant domain.
  • the antibody construct typically includes an immunoglobulin heavy chain disulfide-linked to an immunoglobulin light chain.
  • the antibody construct further may include a third portion bonded to the carboxy terminus of the the second portion comprising a tumoricidal protein.
  • This third protein portion preferably is lymphotoxin, interleukin-2, epidermal growth factor (EGF) , active analogs thereof, or active fragments thereof.
  • the third portion is peptide bonded via an endopeptidase- cleavable amino acid residue, such as lysine, to the carboxy terminus of the CH3 domain of the second region.
  • the third portion may be linked to the second portion by chemical crosslinking, for example.
  • the ⁇ CH2 antibody construct for use as an imaging agent, includes a radioactive label attached thereto.
  • FIG. 1 is a schematic representation showing the construction of a ⁇ CH2 deletion mutant form of the human C ⁇ l gene:
  • A is a map of the Hindlll to PvuII fragment containing the genomic C ⁇ l gene segment and restriction sites;
  • B is the ⁇ CH2 deleted gene missing the Afllll to Avail fragment (the CH2 exon) after restriction and ligation;
  • C shows the Smal site engineered in the CH3 gene;
  • D is a map of a Smal to PvuII linker which is used to attach the (E) PvuII to Xhol fragment containing an IL2 gene;
  • F is is the remainder of the vector including an Xhol site and polyA region;
  • FIG. 2 is a graphic representation of the antigen binding activity of various ⁇ CH2 chimeric constructs: (A) the ⁇ CH2 chl4.18 antibody construct; (B) the ⁇ CH2 chimeric B72.3 (chB72.3) antibody construct;
  • FIG. 3 is a graphic representation of the competitive antigen binding analyses of the intact chl4.12 antibody and the ⁇ CH2 chl4.18 antibody constructs after (A) 2 hours incubation at 37°C; and (B) 18 hours incubation at 4°C;
  • FIG. 4 is a graphic representation of the antibody-dependent cellular cytoxicity (ADCC) of intact antibodies and ⁇ CH2 antibody constructs;
  • FIG. 5 is a graphic representation of the complement-dependent cytotoxicity (CDC) of intact antibodies and ⁇ CH2 antibody constructs
  • FIG. 6 is a graphic representation of the biodistribution of (A) intact chl4.18 antibody and (B) the chl4.18 ⁇ CH2 antibody contruct over time within athymic nude mice bearing M21 xenographic tumors;
  • FIG. 7 is a graphic representation of the biodistribution of F(ab'>2 fragments and the ⁇ CH2 antibody construct within nude mice bearing M21 xenographic tumors.
  • FIG. 8 is a graphic representation of the clearance of the ⁇ CH2 antibody construct, intact antibody, and F(ab')2 fragments from the circulation of athymic nude mice bearing M21 xenographic tumors.
  • the present invention features truncated antibody constructs having specificity for a tumor-associated or other antigen, and demonstrating the in vivo properties of an enhanced binding activity, a relatively short half-life, and a relatively low level of nonspecific binding.
  • ⁇ CH2 constructs are well suited as targeting agents for tumor imaging and systemic pharmacotherapeutic treatment.
  • the invention provides antibody constructs with binding specificity for an epitope on a human tumor antigen.
  • the specificity of the antibody preferably is for a tumor antigen which enables the selective targeting of that tumor, i.e., the construct binds to a unique marker for the tumor.
  • the construct binds to a unique marker for the tumor.
  • antigens include alpha fetoprotein (AFP) and carcinoembryonic antigen (CEA) .
  • Antibody constructs which recognize these or other tumor-associated antigens such as mucin (B72.3 antibody) and the disialoganglioside GQ2 (14.18 antibody) are included in this invention.
  • the ⁇ CH2 constructs of the present invention can be prepared by genetic manipulation of the DNA which encodes the various domains of an antibody (see, e.g.. Neuberger et al. (1984) Nature 312: 604; Morrison et al. (1987) Ann. N.Y. Acd. Sci. 507:187; Morrison et al. (1988) Clin. Chem. 14.:1668; Horwitz et al. (1988) Proc. Natl. Acad. Sci. :8678).
  • the ⁇ CH2 constructs were designed to have a minimal amount of nonhuman protein. This may be accomplished by creating chimeric antibody molecules having at least human constant regions and mammalian, e.g., mouse, variable regions.
  • genomic DNA sequences encoding H and L chains can be cloned in their rearranged forms (i.e., in the DNA sequence that results from recombination events during B Cell maturation) .
  • these genomic sequences contain the information necessary for their expression, (i.e. the 5' untranslated sequences, promoter, enhancer, protein coding region, and donor splice site).
  • the donor splice signals at the 3' end of the V gene segments are compatible with the splice acceptor signals at the 5' end of the Ig regions of other species.
  • the splice product between the two maintains the correct reading frame.
  • mRNA messenger RNA
  • a preferred method of producing the chimeric ⁇ CH2 constructs involves the preparation of an immunoglobulin constant region-encoding cassette including a DNA sequence which enables the splicing of that immunoglobulin constant region-encoding segment to an immunoglobulin variable region-encoding DNA segment having a compatible splice sequence, thereby allowing subsequent transcription and translation of an immunoglobulin heavy or light chain. This method is described in co-pending patent application serial number 409,889 entitiled "Method of Producing Fusion Proteins", filed September 20, 1989, herein incorporated as reference.
  • a DNA cassette is prepared by reconstructing the 3' end of a splice donor site, and its attachment to the 3* end of a DNA sequence or exon encoding a variable (V) region.
  • the cassette is transfected with expressable DNA (structural gene) for a constant (C) region gene having a compatible splice acceptor site at its 5' end.
  • mRNA derived from the two DNA sequences are spliced to produce a mature mRNA having a 5' end encoding a complete V exert or V_ and a 3' end encoding a human C H or C L domain.
  • the resulting single chain polypeptide is a fusion of the V region and the constant domain encoded by the exon of the structural gene.
  • a tumoricidal agent-encoding cassette may be spliced to an Ig C region which, in turn is spliced to an immunoglobulin V region, resulting in a "magic bullet"-type pharmacotherapeutic agent.
  • the cDNA encodes a H or L chain V region (most likely non-human, e.g., murine) specific for a tumor antigen while the C region exon(s) encode the H or L chain C region (CHI, CH2, and CH3 domains) of another (most likely human) Ig species.
  • Useful V regions include the V court and V L domains of murine monoclonal antibody 14.18 (Mujoo et al. (1987) Cancer
  • V regions include the V tensionn and V ⁇ J-i domains of murine monoclonal antibody B72.3 (Johnson et al. (1986) Cancer Res. 4j5:850) which recognize a mucin-like structure on many breast and colon carcinomas. These V region can be combined with various C regions such as that of the Ig human gamma (H) and kappa (L) chains.
  • the V region may be of human origin. Expression of H and L constructs in a single competent host cell results in production of intact chimeric immunoglobulins having a desired specificity and, for example, an intact human constant region.
  • FIG. 1A shows a map of the Hindlll to PvuII fragment containing the human genomic C ⁇ l gene segment.
  • FIG. IB shows the DNA encoding the C region of the ⁇ CH2 construct after restriction and ligation of the two fragments with a synthetic linker (to join the Afllll and Avail ends) .
  • Vectors encoding this mutant C region are transfected into an appropriate host capable of expressing the vectors.
  • the host cell nuclear enzymes produce mRNA by implementing the normal splicing events, resulting in an mRNA encoding a fused protein.
  • the mRNA may encode (5' to 3*) a V sauce or V domain, which may be identical to the native sequence up to the VC junction, attached directly to another polypeptide such as at least a portion of at least one C region domain, which for example, may comprise human sequences.
  • the 3' half of the donor splice site (and any nucleotides downstream) and the 5' half of the acceptor splice site (and any nucleotides upstream) are removed as an intron, resulting in an mRNA encoding the properly fused protein.
  • both exons are placed on the same vector under control of a single regulating sequence. Also, it is preferred to coexpress both L and H chain constructs so that the host cell secretes an intact fusion protein.
  • the method requires use of a host cell having the enzymes which recognize the DNA splice signals and effect proper splicing.
  • vector construction does not, per se, constitute an aspect of the invention, but can be selected and implemented by skilled workers based on personal preference and convenience. Techniques adaptable for use in the invention are disclosed, for example, in Current Protocols in Molecular Biology (Greene Publishing Associates, 430 Fourth Street, Brooklyn, N.Y. , 1989).
  • Useful vectors include any number of known plasmids which contain the correct signals for transcription and translation of the genes of interest. Enhancer elements may be present, and additional signals for polyadenylation and splicing must be present in cases where they are not provided by the gene itself.
  • all of the signals for the expression of functionally rearranged Ig genes are present on a continuous stretch of DNA and include the transcription promoter, the splicing signals for excision of the intron sequences and the polyadenylation and termination sites.
  • Additional information that must be provided by the vector is a selectable marker gene. This gene must also contain the signals for expression of the selectable phenotype (usually resistance to the lethal effect of a toxic drug such as methotrexate, for example) . Therefore, if the vector encodes both the first and second polypeptides, it is necessary that it provide the sequence information for three separate transcription units in a limited amount of space.
  • the recombinant cassette-containing vector is transfected into an appropriate host cell.
  • the choice of host cell line in addition to the criterion noted above, is based on its ability to grow in a growth media, preferably one that is commercially available and serum-free as well as its ease of selectivity after transformation.
  • useful host cells include myelomas or hybridomas such as, for example, the murine non-Ig-producing Sp2/0 Ag 14 hybridoma cell line, yeast, bacteria, and plant cells.
  • Useful host cells are widely available in repositories and from commercial sources and may be isolated readily from natural sources by those skilled in the art.
  • Another transfection method is protoplast (spheroplast) fusion (see, e.g., Sandri-Goldin et al. (1981) Molec. Cell. Biol. 1:743-752).
  • Bacteria harboring the recombinant plasmid of interest are fused to the lymphoid cells with a chemical agent, generally 45-50% polyethylene glycol in a buffered, isotonic solution.
  • This method is simple and does not require extensive purification of plasmid DNA.
  • very high transformation frequencies can be obtained, and the time for obtaining highly productive transfected cell clones is reduced because this transfection method is likely to give transfectants containing multiple copies.
  • guanine phosphoribosyl transferase gpt
  • neomycin neomycin resistance markers
  • the gene encoding the marker would be included on the V-region encoding vector.
  • the resistant form of dihydrofolate reductase DHFR can also be used for the selection of hybridoma cell transformants as well as for subsequent amplification of the marker and flanking product genes.
  • the transfected cell is then cultured to express the polypeptide encoded by the cassette.
  • Culturing may be in. vitro, or in the case of recombinant antibodies, may be accomplished by employing other strategies such as in vivo culturing in ascites fluid.
  • the CH2 domain of the human C ⁇ l heavy chain which is deleted from the intact antibody molecule includes the following sequences: the sequence conferring long half-life; the sequence responsible for binding the Fc receptor on effector cells; the sequence to which the single N-linked carbohydrate chain is attached; and the sequence to which the first complement component, Clq, binds.
  • the ⁇ CH2 constructs were examined closely. When the supernatants of cells expressing the intact and ⁇ CH2 antibodies were first quantitated by measuring associated human K chain to normalize the data to the number of antigen binding domains (antibody molecules contain one K light chain per antigen binding site) , and then compared in a direct antigen binding assay, the ⁇ CH2 constructs demonstrated much higher activity, as shown in FIG. 2. The chl4.18 ⁇ CH2 construct was assayed on G * o2 ⁇ coated plates, while the chB72.3 ⁇ CH2 construct was assayed on mucin-coated plates.
  • the antigen binding activity of the ⁇ CH2 construct was tested further in a competitive binding format in order to rule out the possibility of non-specific interactions with the ELISA plates.
  • the chl4.18 ⁇ CH2 construct competes much more efficiently with the labeled intact chl4.18 antibody for antigen than the normal antibody competes with itself.
  • the length of the binding assay is extended from 2 hours (FIG. 3A) to 18 hours (FIG. 3B) , the difference in binding is not as dramatic, suggesting that the rate of antigen binding and not the overall affinity is increased by the removal of the CH2 domain.
  • the increased antigen binding of ⁇ CH2 antibody constructs reflects conformational changes in the antibody molecule.
  • the CH2 domain By removing the CH2 domain, the Fab region (including both V region, CL and CHI sequences) is no longer restricted by interactions with portions of the CH2 domain. Removal of these inter-domain interactions appears to increase the rate of association with antigen.
  • the removal of the carbohydrate moiety of the CH2 domain may reduce steric interactions between the antibody and the antigen. That the effector functions attributed to the CH2 domain are no longer retained in the ⁇ CH2 antibody can be confirmed by ADCC assay using the intact chl4.18 antibody as a basis for comparison.
  • FIG. 4 The results of a typical ADCC assay, which measures the ability of unfractionated peripheral blood leukocytes (PBLS) to lyse melanoma target cells as a function of the amount of added antibody, are shown in FIG. 4.
  • PBLS peripheral blood leukocytes
  • FIG. 4 the specific lysis of M21 human melanoma cells in the presence of the indicated concentrations of chl4.18 antibody or ⁇ CH2 construct is shown for an effector to target ratio of 100:1.
  • the average of triplicate samples is shown for each data point.
  • the intact chl4.18 antibody is a very potent mediator of ADCC, even at levels as low as 1 ng/ml.
  • the ⁇ CH2 construct exhibits " a very small amount of ADCC activity.
  • the loss of ADCC activity also reflects a loss of the ability to bind to Fc receptors. Since the site for binding of the high-affinity receptor (FCR) has been mapped to the CH2 domain, adjacent to the hinge, it is not surprising that the ⁇ CH2 mutant had greatly reduced activity. For the radioactive imaging of tumors, it is important that Fc receptor binding be reduced so that there is minimal nonspecific accumulation in the spleen, where many Fc-receptor-bearing cells localize. The ⁇ CH2 construct here described is therefore useful in this regard.
  • FIG. 5 shows that the ⁇ CH2 mutant had no measurable ability to lyse M21 cells in the presence of human complement relative to the very potent chl4.18 antibody.
  • Anti-tumor ⁇ CH2 antibodies having the ability to specifically and rapidly localize to the tumor, also serve as ideal vehicles for the delivery of therapeutic agents.
  • the DNA sequence encoding human interleukin-2 (IL2) may be attached to the carboxy terminus of an anti-tumor ⁇ CH2 construct, thereby resulting in the expression of a ⁇ CH2/IL2 fusion protein that serves to deliver IL2 to the tumor site.
  • the local accumulation of IL2 activates T-cells at the tumor site, thereby hastening its destruction.
  • the half-life of ⁇ CH2 antibodies is shorter than whole antibodies, the ⁇ CH2/IL2 fusion protein should be much longer than the half-life of IL2.
  • the anti-tumor cytokine lymphotoxin or tumor necrosis factor ⁇ could be fused to the carboxy terminus of a ⁇ CH2 antibody.
  • This fusion protein also serves to specifically localize this cytotoxic protein to a tumor, thus reducing its potential adverse systemic effects.
  • other toxic proteins could be fused to ⁇ CH2 antibodies. These include protein toxins such as ricin, diphtheria toxin, and Pseudomona exotoxin or those portions of the whole toxin molecule that is responsible for toxicity, i.e. the ADP-ribosylating enzyme activity that leads to the inactivation of mammalian cell elongation factor 2.
  • FIG. 6 and TABLE 1 show the biodistribution of (A) intact chl4.18 antibody and (B) a ⁇ CH2 construct with time, and demonstrates that the latter targets quickly (within 4 hours) and specifically to the tumor.
  • FIG. 7 shows the biodistribution of human Ig F(ab*)2 fragments and chl4.18 ⁇ CH2 constructs 24 hours after injection, and demonstrates the specificity of targeting of the ⁇ CH2 constructs.
  • the half-life of the ⁇ CH2 antibody constructs in circulation may be determined by injecting them into the circulation of tumor-bearing animals and monitoring their presence in the blood with time.
  • FIG. 8 demonstrates the short half-life of ⁇ CH2 constructs relative to F(ab')2 fragments and intact antibodies in the circulation of nude mice bearing M21 xenographic tumors.
  • the ⁇ CH2 antibody constructs are able to bind quickly to a tumor epitope with relatively little non-specific binding to other tissues, they can be used to target various therapeutic agents to tumors.
  • Particularly useful therapeutic agents include those having direct or indirect tumoricidal activity such as interleukin-2, lymphotoxin, or epidermal growth factor.
  • EGF is fused to an antibody that binds to and activates a cytotoxic T-cell, thus cross-linking the T cell and EGF receptor-bearing cell. Fragments and biosynthetic analogs of these proteins having tumoricidal activity also are useful.
  • These proteins may be chemically linked to the CH3 domain via, for example, various cross-linking agents known in the art, or, as disclosed herein, may be peptide-bonded to the carboxy terminus of the CH3 domain.
  • the vectors constructed to encode the ⁇ CH2 constructs may further encode the tumoricidal protein.
  • the increased binding activity of the ⁇ CH2 construct together with its shorter half-life in vivo, and its low level of nonspecific binding make this molecule extremely useful in the radioactive imaging and/or treatment of tumors.
  • the constructs may be labeled with, for example, radionuclides of low energy such as iodine-123, indium-Ill, or technetium-99m, or with any radioactive isotopes that will not change the binding characteristics of the construct when attached thereto, such as iodine-125.
  • radionuclides of low energy such as iodine-123, indium-Ill, or technetium-99m
  • any radioactive isotopes that will not change the binding characteristics of the construct when attached thereto, such as iodine-125.
  • the ⁇ CH2 antibody constructs enable detection of the locus of a tumor-related antigen by external detection of emitted radiation.
  • Radioactive gamma emitters are preferred, although in some circumstances positron emitters, such as 64 Cu, 11 C, and 15 0 may be used. It is also contemplated that the ⁇ CH2 antibody constructs may be labelled with a paramagnetic substance such as gadolinium so that concentrations of the reagent localized about a tumor may be imaged by nuclear magnetic resonance imaging techniques.
  • Targeting of a construct with a tumoricidal agent and/or radionuclide may require a higher concentration of construct having a longer half-life than does imaging.
  • the level of radioactivity required for imaging depends in part on the ability of the antibody construct to selectively label tumor relative to surrounding tissue, the size of the tumor, and the distance of the tumor from the injection site.
  • the ⁇ CH2 antibody constructs of the invention can be labelled with such agents using conventional techniques used to label other proteins known to those skilled in the art.
  • One currently preferred technique is to express a ⁇ CH2 antibody construct, covalently linked on either its 3' or 5' end to a lysine rich polypeptide sequence comprising, for example, 2-20 residues, to serve as a site of attachment for the remotely detectable moieties discussed above.
  • the gamma or positron emitting ions are preferably attached to these linkers by ionic interaction or chelation with the free amine groups on the lysine residues.
  • diethylenetriamine penta acetic acid may be used to label with Tc-99 or In-Ill.
  • In or I isotopes may be attached using the oxine salt (8-hydroxy quinoline) .
  • Iodogen (1, 3, 4, 6 tetrachloro - 3 ⁇ , 6 ⁇ diphenylglucoluril - Pierce Chem. Co., Roskville, 111) may be used for labeling with 125 I.
  • Other methods are known to those skilled in the art.
  • reagents can be linked using conventional techniques to ⁇ CH2 antibody contructs.
  • therapeutic drugs which promote healings such as calcitonin, epidermal growth factor, tumor growth factor alpha and beta, platelet derived growth factor, insulin-like growth factor 1 (Somatomedin-C), connective tissue activating peptide, and human collagenase inhibitor.
  • the dosage and means of administration of the family of ⁇ CH2 antibody contructs produced in accordance with the invention will necessarily depend on the nature of the drug involved, the degree if any that its bioactivity is reduced when it is present as a conjugate, the immunological tolerance of specific patients, and the nature of the diseased condition in question.
  • the polypeptide When the polypeptide is linked to an imaging agent, it should be administered in an amount sufficient to be detected by scintillation scanning or other external radiation detection means capable of detecting the localized radiation present in the tumor, such as autoradiography ⁇
  • about 5-20 microcuries should be administered; most preferably about 10 microcuries. The actual amount depends on the location of the tumor-associated antigen as is well known in the art. Additional radioactive peptide may be injected if necessary up to the amounts limited by prevalent standards of safety.
  • Tumors may be imaged or treated by systemic administration of the ⁇ CH2 construct via intravenous injection. Their preferred dosage is in the range of from about 0.01 to 10 mg per kg body weight.
  • the human C ⁇ l gene subcloned in pBR322 as a Hindlll to PvuII fragment (Gillies et al. (1989) J. Immuno. Meth. 125:191-202) was used for the construction of the H chain mutants.
  • the deletion mutant lacking the CH2 domain ( ⁇ CH2) was made by joining the Hindlll to Afllll fragment (containing the CHI and hinge exons) to an Avail to PvuII fragment (containing the CH3 exon) using a synthetic double-stranded oligonucleotide fragment.
  • the ⁇ CH2 construct was checked by DNA sequence analysis and then introduced into the chimeric antibody expression vector pdHL2-VC ⁇ lC ⁇ (Gillies et al. ibid.) containing the V regions of the mouse monoclonal antibody 14.18 (Mujoo et al. (1987) Cancer Res. 47:1908) .
  • a ⁇ CH2-IL2 fusion construct was constructed by first introducing a Smal site by mutation near the carboxy terminus of the ⁇ CH2 human IgGl gene (FIG. 1(C). The mutation changes nucleotide 2373 (according to the numbering in the Huck et al., (1986) Nucl. Acids. Res. 11:1779-1789) from a T to a C in the wobble position of a serine codon and thus does not change the amino acid sequence. A synthetic DNA fragment was used to link the mature IL2 protein sequence to this Smal site (FIG. ID-IE) .
  • the sequence includes, from 5* to 3', the Smal site, the remaining sequence of the human gamma 1 gene (three additional amino acids) fused directly to the first amino acid (alanine) of the mature IL2 protein sequence, and the amino terminal sequence of IL2 up to the unique PvuII site.
  • the remaining IL2 sequence was then joined at the PvuII site and extends through the coding sequence to a unique Xhol site located 3* of the translational stop signal.
  • the Xhol end of the joined DNA was ligated to a vector containing the 3' untranslated region and polyA addition site of the mouse K constant region (FIG. IF) .
  • the resulting construct encodes a ⁇ CH2 human gamma 1 chain to which IL2 is attached at the carboxy terminus.
  • Transfection and drug selection were carried out essentially as described in Gillies et al. (Bio/Technol. (1989) 2:799-804), herein incorporated as reference. Briefly, the non-Ig-producing murine hybridoma line, Sp2/0 Agl4, was maintained in
  • DMEM Dulbecco's modified Eagle's medium
  • Plasmids were introduced into cells by protoplast fusion essentially as described in Gillies et al., (1983) Cell 22: 717-728) .
  • Transfectants were selected in growth medium containing methotrexate at an initial concentration of 0.1 ⁇ M.
  • MTX-resistant clones were tested for the secretion of human antibody by ELISA assay.
  • Microtiter plates were coated with goat anti-human IgG (H and L specific-Jackson
  • the chl4.18 intact antibodies were purified by binding to and elution from protein A Sepharose (Repligen) .
  • the ⁇ CH2 mutant antibody was purified by immunoaffinity chromatography on an anti-human K monoclonal antibody-Sepharose column. Both proteins were >90% pure when analyzed by SDS-PAGE or HPLC. 4. Antigen Binding Assays
  • Direct antigen binding assays as well as competitive binding assays were performed with the disialoganglioside GD2 antigen (14.18 antibody) or submaxillary gland mucin (Sigma) (B72.3 antibody)- coated microtiter plates as described in Gillies et al. (1990, ibid.) .
  • the secondary (detecting) antibody was a goat anti-human IgG, Fc-specific polyclonal antiserum (Jackson
  • ADCC assays were carried out using 51 Cr- labeled M21 human melanoma target cells or any cell line which expresses GD2-
  • a fixed number of labeled targets (2 x IO 5 cells/ml in 50 ⁇ L) and varying concentrations of human effector cells (peripheral blood leukocytes from normal donors) in 50 ⁇ L were mixed with 100 ⁇ L of diluted chimeric antibodies in round-bottom microtiter plates. Following a 4 hr incubation at 37°C, the plates were centrifuged, and 100 ⁇ L samples were removed for counting in a LKB model 1272 gamma counter.
  • Complement-mediated lysis of labeled M21 melanoma cells was carried out by mixing 50 ⁇ L of cells (2 x 10 s cells/mL) with an equal volume of each antibody dilution. After a 15 min incubation at 37°C, 100 ⁇ L of human complement (1:4 dilution of fresh human serum) was added to each well. Plates were incubated for an additional hour at 37°C, and the amount of released 51 Cr was determined by centrifuging the plates and counting 100 ⁇ L aliquots of the supernatants. The percentage of cytotoxicity was determined as:
  • mice 8 to 10 weeks old female athymic mice (nu/nu) (the National Cancer Institute, Bethesda, MD) were injected subcutaneously with 2 x IO 6 M21 tumor cells. Tumors of 50-150 mg weight grew within 10 days. At this time, the animals received i.v. injections into the lateral tail vein of 25 ⁇ g and 3-4 ⁇ Ci 125 I-labeled antibody. At designated time points after injection, groups of 3 animals were anesthetized with halothane, and blood samples were obtained by retro-orbital bleeding. For biodistribution of radiolabeled antibodies, groups of 3 animals were sacrificed at various time points after injection. Tumors and major organs (heart.
  • tissue samples were assayed in a gamma counter for 125 I activity. The results were calculated as percent of injected dose per g tissue and as localization ratios (cpm/g tumor : cpm/g tissue) .
  • Chl4.18, chl4.18- ⁇ CH2, and F(ab') 2 fragments of human IgG were labeled with 125 I. Briefly, 500 ⁇ g antibody was incubated for 25 min on ice with 0.5 mCi 125 I (100 mCi or 3.75 GBq/ml, Amersham Corp., Arlington Heights, IL) in polystyrene tubes coated with 100 ⁇ g Iodo-Gen reagent (Pierce Chemical Co., Rockford, IL) . Unincorporated 125 I was removed by gel filtration on PD10 columns (Pharmacia Fine Chemicals, Piscataway, NJ) . Specific activity was typically 1.5 - 0.5 nCI/ng antibody.

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Abstract

Construction d'anticorps ayant une activité de liaison améliorée spécifique à un déterminant antigénique sur un antigène. Ladite construction comprend une région de liaison de l'immunoglobuline de spécificité prédéterminée et ayant plusieurs régions hypervariables identiques à une région hypervariable d'un anticorps non dénaturé spécifique audit déterminant antigénique. Ladite construction comprend en outre une région de constance de l'immunoglobuline consistant essentiellement en un domaine CH1, une charnière et un domaine CH3 et omettant le domaine CH2 normalement présent dans un anticorps non dénaturé. La région de constance de l'immunoglobuline améliore au moins deux fois l'activité de liaison en comparaison à l'activité de liaison d'un anticorps non dénaturé ayant la même spécificité.
PCT/US1991/000633 1990-03-02 1991-01-30 Constructions d'anticorps ayant une affinite de liaison amelioree WO1991013166A1 (fr)

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Cited By (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992008495A1 (fr) * 1990-11-09 1992-05-29 Abbott Biotech, Inc. Immunoconjugues de cytokine
US5650150A (en) * 1990-11-09 1997-07-22 Gillies; Stephen D. Recombinant antibody cytokine fusion proteins
WO2002072605A3 (fr) * 2001-03-07 2002-11-14 Lexigen Pharm Corp Technique d'expression pour des proteines contenant un fragment d'anticorps isotype chimerique
US6617135B1 (en) 1999-08-09 2003-09-09 Emd Lexigen Research Center Corp. Multiple cytokine protein complexes
US6838260B2 (en) 1997-12-08 2005-01-04 Emd Lexigen Research Center Corp. Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
US6969517B2 (en) 2001-05-03 2005-11-29 Emd Lexigen Research Center Corp. Recombinant tumor specific antibody and use thereof
US6992174B2 (en) 2001-03-30 2006-01-31 Emd Lexigen Research Center Corp. Reducing the immunogenicity of fusion proteins
US7026446B1 (en) * 1997-12-24 2006-04-11 Diatech Pty Ltd. Bifunctional molecules
EP1663306A2 (fr) * 2003-09-05 2006-06-07 Genentech, Inc. Anticorps aux fonctions d'effecteur modifiees
US7067110B1 (en) 1999-07-21 2006-06-27 Emd Lexigen Research Center Corp. Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens
US7091321B2 (en) 2000-02-11 2006-08-15 Emd Lexigen Research Center Corp. Enhancing the circulating half-life of antibody-based fusion proteins
US7169904B2 (en) 2002-12-17 2007-01-30 Emd Lexigen Research Center Corp. Immunocytokine sequences and uses thereof
US7186804B2 (en) 2001-12-04 2007-03-06 Emd Lexigen Research Center Corp. IL-2 fusion proteins with modulated selectivity
US7211253B1 (en) 1999-11-12 2007-05-01 Merck Patentgesellschaft Mit Beschrankter Haftung Erythropoietin forms with improved properties
US7323549B2 (en) 2003-12-30 2008-01-29 Emd Lexigen Research Center Corp. IL-7 fusion proteins
US7432357B2 (en) 2004-01-22 2008-10-07 Merck Patent Gmbh Anti-cancer antibodies with reduced complement fixation
US7465447B2 (en) 2003-12-31 2008-12-16 Merck Patent Gmbh Fc-erythropoietin fusion protein with improved pharmacokinetics
US7517526B2 (en) 2000-06-29 2009-04-14 Merck Patent Gmbh Enhancement of antibody-cytokine fusion protein mediated immune responses by combined treatment with immunocytokine uptake enhancing agents
US7589179B2 (en) 2004-12-09 2009-09-15 Merck Patent Gmbh IL-7 variants with reduced immunogenicity
US8106161B2 (en) 2001-01-17 2012-01-31 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
US8147835B2 (en) 2001-01-17 2012-04-03 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
US8333966B2 (en) 2008-04-11 2012-12-18 Emergent Product Development Seattle, Llc CD37 immunotherapeutics and uses thereof
US8409577B2 (en) 2006-06-12 2013-04-02 Emergent Product Development Seattle, Llc Single chain multivalent binding proteins with effector function
US8691952B2 (en) 2005-12-30 2014-04-08 Merck Patent Gmbh Anti-CD19 antibodies with reduced immunogenicity
US8907066B2 (en) 2009-04-22 2014-12-09 Merck Patent Gmbh Antibody fusion proteins with a modified FcRn binding site
US9029330B2 (en) 2005-12-30 2015-05-12 Merck Patent Gmbh Methods of treating cancer using interleukin-12p40 variants having improved stability
US10143748B2 (en) 2005-07-25 2018-12-04 Aptevo Research And Development Llc B-cell reduction using CD37-specific and CD20-specific binding molecules
US10604576B2 (en) 2016-06-20 2020-03-31 Kymab Limited Antibodies and immunocytokines
US11352426B2 (en) 2015-09-21 2022-06-07 Aptevo Research And Development Llc CD3 binding polypeptides
US11753479B2 (en) 2014-03-04 2023-09-12 Kymab Limited Nucleic acids encoding anti-OX40L antibodies
US11779604B2 (en) 2016-11-03 2023-10-10 Kymab Limited Antibodies, combinations comprising antibodies, biomarkers, uses and methods
US12209128B2 (en) 2016-06-20 2025-01-28 Kymab Limited Anti-PD-L1 antibodies

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EP0256654A2 (fr) * 1986-07-07 1988-02-24 Centocor, Inc. Immunoglobuline chimère murine-humaine, spécifique pour l' antigène 17-1A associés aux tumeurs

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US5650150A (en) * 1990-11-09 1997-07-22 Gillies; Stephen D. Recombinant antibody cytokine fusion proteins
EP1149913A1 (fr) * 1990-11-09 2001-10-31 GILLIES, Stephen D. Immunoconjugués de cytokine
WO1992008495A1 (fr) * 1990-11-09 1992-05-29 Abbott Biotech, Inc. Immunoconjugues de cytokine
US6838260B2 (en) 1997-12-08 2005-01-04 Emd Lexigen Research Center Corp. Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
US7226998B2 (en) 1997-12-08 2007-06-05 Emd Lexigen Research Center Corp. Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
US7576193B2 (en) 1997-12-08 2009-08-18 Merck Patent Gmbh Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
US7026446B1 (en) * 1997-12-24 2006-04-11 Diatech Pty Ltd. Bifunctional molecules
US7067110B1 (en) 1999-07-21 2006-06-27 Emd Lexigen Research Center Corp. Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens
US7141651B2 (en) 1999-08-09 2006-11-28 Emd Lexigen Research Center Corp. Multiple cytokine protein complexes
US6617135B1 (en) 1999-08-09 2003-09-09 Emd Lexigen Research Center Corp. Multiple cytokine protein complexes
US7582288B2 (en) 1999-08-09 2009-09-01 Merck Patent Gmbh Methods of targeting multiple cytokines
US7211253B1 (en) 1999-11-12 2007-05-01 Merck Patentgesellschaft Mit Beschrankter Haftung Erythropoietin forms with improved properties
US7507406B2 (en) 2000-02-11 2009-03-24 Emd Serono Research Center, Inc. Enhancing the circulating half-life of antibody-based fusion proteins
US7091321B2 (en) 2000-02-11 2006-08-15 Emd Lexigen Research Center Corp. Enhancing the circulating half-life of antibody-based fusion proteins
US7517526B2 (en) 2000-06-29 2009-04-14 Merck Patent Gmbh Enhancement of antibody-cytokine fusion protein mediated immune responses by combined treatment with immunocytokine uptake enhancing agents
US9005612B2 (en) 2001-01-17 2015-04-14 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
US8188237B2 (en) 2001-01-17 2012-05-29 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
US8197810B2 (en) 2001-01-17 2012-06-12 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
US8147835B2 (en) 2001-01-17 2012-04-03 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
US8106161B2 (en) 2001-01-17 2012-01-31 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
US8853366B2 (en) 2001-01-17 2014-10-07 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
CN1330664C (zh) * 2001-03-07 2007-08-08 默克专利有限公司 用于含杂合同种型抗体部分的蛋白质的表达技术
US8066994B2 (en) 2001-03-07 2011-11-29 Merck Patent Gmbh Proteins comprising an IgG2 domain
US7148321B2 (en) 2001-03-07 2006-12-12 Emd Lexigen Research Center Corp. Expression technology for proteins containing a hybrid isotype antibody moiety
WO2002072605A3 (fr) * 2001-03-07 2002-11-14 Lexigen Pharm Corp Technique d'expression pour des proteines contenant un fragment d'anticorps isotype chimerique
US8926973B2 (en) 2001-03-30 2015-01-06 Merck Patent Gmbh Reducing the immunogenicity of fusion proteins
US6992174B2 (en) 2001-03-30 2006-01-31 Emd Lexigen Research Center Corp. Reducing the immunogenicity of fusion proteins
US7601814B2 (en) 2001-03-30 2009-10-13 Merck Patent Gmbh Reducing the immunogenicity of fusion proteins
US7459538B2 (en) 2001-05-03 2008-12-02 Merck Patent Gmbh Recombinant tumor specific antibody and use thereof
US6969517B2 (en) 2001-05-03 2005-11-29 Emd Lexigen Research Center Corp. Recombinant tumor specific antibody and use thereof
US7186804B2 (en) 2001-12-04 2007-03-06 Emd Lexigen Research Center Corp. IL-2 fusion proteins with modulated selectivity
US7462350B2 (en) 2001-12-04 2008-12-09 Emd Serono Research Center, Inc. Cancer treatments including administering IL-2 fusion proteins with modulated selectivity
US7169904B2 (en) 2002-12-17 2007-01-30 Emd Lexigen Research Center Corp. Immunocytokine sequences and uses thereof
EP1663306A2 (fr) * 2003-09-05 2006-06-07 Genentech, Inc. Anticorps aux fonctions d'effecteur modifiees
US7323549B2 (en) 2003-12-30 2008-01-29 Emd Lexigen Research Center Corp. IL-7 fusion proteins
US7465447B2 (en) 2003-12-31 2008-12-16 Merck Patent Gmbh Fc-erythropoietin fusion protein with improved pharmacokinetics
US7432357B2 (en) 2004-01-22 2008-10-07 Merck Patent Gmbh Anti-cancer antibodies with reduced complement fixation
US10633452B2 (en) 2004-01-22 2020-04-28 Merck Patent Gmbh Anti-cancer antibodies with reduced complement fixation
US10017579B2 (en) 2004-01-22 2018-07-10 Meck Patent Gmbh Anti-cancer antibodies with reduced complement fixation
US8835606B2 (en) 2004-01-22 2014-09-16 Merck Patent Gmbh Anti-cancer antibodies with reduced complement fixation
US9617349B2 (en) 2004-01-22 2017-04-11 Merck Patent Gmbh Anti-cancer antibodies with reduced complement fixation
US7589179B2 (en) 2004-12-09 2009-09-15 Merck Patent Gmbh IL-7 variants with reduced immunogenicity
US10307481B2 (en) 2005-07-25 2019-06-04 Aptevo Research And Development Llc CD37 immunotherapeutics and uses thereof
US10143748B2 (en) 2005-07-25 2018-12-04 Aptevo Research And Development Llc B-cell reduction using CD37-specific and CD20-specific binding molecules
US11208496B2 (en) 2005-12-30 2021-12-28 Cancer Research Technology Ltd. Anti-CD19 antibodies with reduced immunogenicity
US9029330B2 (en) 2005-12-30 2015-05-12 Merck Patent Gmbh Methods of treating cancer using interleukin-12p40 variants having improved stability
US8691952B2 (en) 2005-12-30 2014-04-08 Merck Patent Gmbh Anti-CD19 antibodies with reduced immunogenicity
US10072092B2 (en) 2005-12-30 2018-09-11 Merck Patent Gmbh Methods of use of anti-CD19 antibodies with reduced immunogenicity
US8957195B2 (en) 2005-12-30 2015-02-17 Merck Patent Gmbh Anti-CD19 antibodies with reduced immunogenicity
US8409577B2 (en) 2006-06-12 2013-04-02 Emergent Product Development Seattle, Llc Single chain multivalent binding proteins with effector function
US9101609B2 (en) 2008-04-11 2015-08-11 Emergent Product Development Seattle, Llc CD37 immunotherapeutic and combination with bifunctional chemotherapeutic thereof
US8333966B2 (en) 2008-04-11 2012-12-18 Emergent Product Development Seattle, Llc CD37 immunotherapeutics and uses thereof
US8907066B2 (en) 2009-04-22 2014-12-09 Merck Patent Gmbh Antibody fusion proteins with a modified FcRn binding site
US11753479B2 (en) 2014-03-04 2023-09-12 Kymab Limited Nucleic acids encoding anti-OX40L antibodies
US11773175B2 (en) 2014-03-04 2023-10-03 Kymab Limited Antibodies, uses and methods
US11352426B2 (en) 2015-09-21 2022-06-07 Aptevo Research And Development Llc CD3 binding polypeptides
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