CA2077348A1 - Antibody constructs with enhanced binding affinity - Google Patents
Antibody constructs with enhanced binding affinityInfo
- Publication number
- CA2077348A1 CA2077348A1 CA002077348A CA2077348A CA2077348A1 CA 2077348 A1 CA2077348 A1 CA 2077348A1 CA 002077348 A CA002077348 A CA 002077348A CA 2077348 A CA2077348 A CA 2077348A CA 2077348 A1 CA2077348 A1 CA 2077348A1
- Authority
- CA
- Canada
- Prior art keywords
- construct
- antibody
- domain
- binding
- region
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- 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
Abstract
Disclosed is an antibody construct with enhanced binding activity specific for an epitope on an antigen. The construct includes an immunoglobulin binding region of predetermined specificity and having plural hypervariable regions homologous with a hypervariable region of a native antibody specific for the epitope. The construct further includes an immunoglobulin constant region consisting essentially of a CH1 domain, a hinge, and a CH3 domain and omitting the CH2 domain normally present in a native antibody. The immunoglobulin constant region enhances the binding activity of the construct at least two fold in comparison with the binding activity of a native antibody having the same specifity.
Description
,.0 91/13166 PC~r/US91/00633 -'- 2 0 7 7 .~
ANTIBODY CONSTRUCTS WITH ENHANCED BINDING AFFINITY ~
~ . .
5 Backqround o~ the Invention -This invention relates to cancér imaging --~
agents and cancer therapeutic agents, and more specifically, to the preparation of antibody 10 constructs useful as imaging agents and/or cancer therapeutic agents. In particular, 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. Likewise is the administration of ;'~
labelling agents such as radioisotopes for tumor or -organ imaging. However, the administration of such -20 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 25 the ability to interere 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 30 type.
Antibody molecules have been conjugated to ~;~ various drugs, enzymes, toxins, and radioisotopes (reviewed in Ghose and Bla~r (1~78) J. Natl. Cancer .
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Inst. 61:657-676). For example, antibodies have been successfully employed to image occult tumors not detected by traditional detection methods such as magnetic resonance imaging, x-ray analysis, and 5 computed tomography (see Goldenberg (1988) Arch.
Pathol. Lab. Med. 112:580-587).
However, a number of problems have been associated with the use of antibody molecules as 10 targeting agents. 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.). In addition, there may be a high level of nonspecific binding of the intact 15 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. Furthermore, intact antibodies have 20 a slow ~learance rate relative to smaller molecules such as fragments of antibody molecules.
In an effort to all~viate these problems, the prior art suggests the use of antibody fragments 25 which maintain their specificity and ability to bind ~ --antigen. Antibody fragments display more rapid specific targeting than intact antibodies. Wahl et al. (1983) J. Nuclear Med. 24:317-325). Currently, enzymatically produced F(ab')2 fragments are most 30 commonly used for such applications. These fragments lack the potentially troublesome Fc portion of the ~-intact antibody molecule, and so demonstrate lower -non-specific accumulation in the liver and spleen and a faster rate of clearance than intact antibodies.
` ~ O 91/13166 P ~ /~'S91/00633 ;
ANTIBODY CONSTRUCTS WITH ENHANCED BINDING AFFINITY ~
~ . .
5 Backqround o~ the Invention -This invention relates to cancér imaging --~
agents and cancer therapeutic agents, and more specifically, to the preparation of antibody 10 constructs useful as imaging agents and/or cancer therapeutic agents. In particular, 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. Likewise is the administration of ;'~
labelling agents such as radioisotopes for tumor or -organ imaging. However, the administration of such -20 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 25 the ability to interere 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 30 type.
Antibody molecules have been conjugated to ~;~ various drugs, enzymes, toxins, and radioisotopes (reviewed in Ghose and Bla~r (1~78) J. Natl. Cancer .
'~ .
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.O 91/13166 P ~ /US91/00633 -2- 2077~
Inst. 61:657-676). For example, antibodies have been successfully employed to image occult tumors not detected by traditional detection methods such as magnetic resonance imaging, x-ray analysis, and 5 computed tomography (see Goldenberg (1988) Arch.
Pathol. Lab. Med. 112:580-587).
However, a number of problems have been associated with the use of antibody molecules as 10 targeting agents. 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.). In addition, there may be a high level of nonspecific binding of the intact 15 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. Furthermore, intact antibodies have 20 a slow ~learance rate relative to smaller molecules such as fragments of antibody molecules.
In an effort to all~viate these problems, the prior art suggests the use of antibody fragments 25 which maintain their specificity and ability to bind ~ --antigen. Antibody fragments display more rapid specific targeting than intact antibodies. Wahl et al. (1983) J. Nuclear Med. 24:317-325). Currently, enzymatically produced F(ab')2 fragments are most 30 commonly used for such applications. These fragments lack the potentially troublesome Fc portion of the ~-intact antibody molecule, and so demonstrate lower -non-specific accumulation in the liver and spleen and a faster rate of clearance than intact antibodies.
` ~ O 91/13166 P ~ /~'S91/00633 ;
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2~77348 - -For example, several days after the administration of radiolabelled, intact antibody IgG, the level of circulating and nonspecifically bound radioactivity is high, and clearance occurs gradually over a period 5 of four days or more (Goldenberg, ibid.). In -comparison, 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 123I and 99mTc (Goldenberg, 10 ibid.).
However, the use of F(ab')2 fragments is not without pitfalls. Besides being difficult to produce enzymatically, the binding properties of these 15 fragments may be compromised (~ç~, e.a., Wahl et al., ibid. Therefore, what is needed is a targeting molecule ~aving specificity, enhanced binding activity, minimal nonspecific binding abilities, and a shorter half-life in vivo.
Towards this end, monoclonal antibodies with increased antigen binding activites have been identified which have mUtationS in their constant regions ~see, e.g., Pollock et al. (1988) Proc. Natl.
25 Acad. Sci. USA 85:2298-2302). In addition, the ability to genetically engineer and re-express -antibodies in transfected cells has made it possible to combine various portions of the immunoglobulin molecule as fusion proteins (Neuberger et al. (1984) , 30 Nature 312:614; Morrison et al. (1987~ Ann. N.Y.
Acad. Sci. 507:187; and Morrison et al. (1988) Clin. ;
Chem. 34:1668). It is also possible to genetically - encode immunoglobulin deletion mutants such as Fab (Horwitz et al. (1988) Proc. Natl. Acad. Sci. U.S.A. -",~
' .O9l/13166 PCT/US91/00633 2077~18 85:8687) or F(ab')2-like fragments. In one study, an Fd~ fragment, a truncated form of the immunoglobulin heavy chain, was expressed in E. coli (Cabilly et al.
(1984) Proc. Natl. Acad. Sci. USA 81:3273-3277). In 5 another study, specific constant region domains of the human Cy3 H chain were either deleted or added in order to assess the effect on antibody synthesis and assembly (Morrison et al. (1987) Ann. N.Y. Acad. Sci.
507:1873).
Accordingly, it is 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 15 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 oS the body is reduced. Additional 20 ob~ects 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.
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summary of the Invention It has been discovered that antibody constructs retaining their CHl and CH3 domains, but 5 lacking their CH2 domain, surprisingly have an enhanced binding affinity as well as a shorter half-life and an unexpectedly low level of non-specific binding relative to an intact antibody molécule with the same specificity. This finding has 10 been exploited to develop the present invention which features antibody constructs having specificity for an epitope, present on various tumor antigens.
Native antibody molecules of the IgG and IgD
15 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 (~) domains 2t the amino termini of the two chains together form the antigen binding region. The 20 first constant ~C) domain of the H chain (CH1) interacts with the C region of the L chain through hydrophobic interactions and also through a disulfide bond. The ne~t H-chàin domain, adjacent to the hinge, is called CH2. This domain reportedly 25 contains many o the effector functions of the antibody including the sequences responsible or complement fixation and Fc receptor-mediated -antibody-dependent cellular cytotoxicity (ADCC), and is the sole N-linked glycosylation site in human Cyl 30 chains. The CH3 domain at the carboxy terminus of the H-chain is thought to play a major role in the assembly of H chains. :
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:_0 91tl3166 PCI/US91/00633 2077~48 The antibody constructs of the present invention include a first portion having an immunoglobulin binding region of predetermined specificity. This binding region has plural 5 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 CHl domain and a hinge whose C terminus is linked to the N
10 terminus of a CH3 domain, i.e., a hybrid constant domain which omits the CH2 domain normally present in a native antibody. ~.ntibody constructs with the CH2 domain deleted are referred to hereinafter as ~CH2 constructs. ~CH2 constructs are characterized by 15 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.
. .
In one preferred embodiment, the binding region of the ~CH2 antibody construct includes the binding region of a nonhuman immunoglobulin such as a murine immunoglobulin. In one aspect o~ the invention, the immunoglobulin binding region is-one 25 which has a predetermined specificity for mucin such - -as the binding region of monoclonal antibody B72.3, or has a predetermined specificity for the GD2 - -ganglioside such as the binding region of monoclonal antibody 14.18. In another embodiment of the 30 invention, the immunoglobulin binding region has a predetermined specificity for a melanoma-specific proteoglycan. The preferred altered constant region - -- includes a human immunoglobulin constant domain.
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_7_ 2Q77~`~18 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 carbo~y terminus of the the second portion comprising a tumoricidal protein.
This third protein portion preferably is lymphotoxin, interleukin-2, epidermal growth factor ~EGF), active lO analogs thereof, or active fragments thereof. In a preferred embodiment of the invention, the third portion is peptide bonded via an endopeptidase-cleavable amino acid residue, such as lysine, to the carbo~y terminus of the CH3- domain of the second 15 region. Alternatively, the third portion may be linked to the second portion by chemical crosslinking, for esample.
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In yet another embodiment, for use as an 20 imaging agent, the ~CH2 antibody construct includes a radioactive label attached thereto.
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~.091tl3166 PCI/I,IS91/00633 20773~8 Brief Description of the Drawing The foregoing and other objects of the -present invention, the various features thereof, as 5 well as the inventions thereof may be more ~ully ~ ~
understood from the following description when read ~ --together with the accompanying drawings in which:
FIG. 1 is a schematic representation showing 10 the construction of a ~CH2 deletion mutant form of , the hu~an Cyl gene: (A) is a map of the HindIII to PvuII fragment containing the genomic Cyl gene segment and restriction sites; (B) is the ~CH2 deleted gene missing the AflIII to AvaII fragment 15 (the CH2 exon) after restriction and ligation; (C) shows the SmaI site engineered in the CH3 gene; (D) is a map of a SmaI to PvuII linker which is used to ~
at~ach the (E) PvuII to XhoI fragment containing an :
I~2 gene; and ~F) is is the remainder of the vector 20 including an XhoI site and polyA region;
, FIG. 2 is a graphic representation of the antigen binding activity of variaus ~C~2 chimeric constructs: (A) ~he ~CH2 chl4.18 antibody construct;
25 (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 30 chl4.12 antibody and the ~CH2 chl4.18 antibody constructs after (A) 2 hours incubation at 37C; and (B) 18 hours incubation at 4C;
.
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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 lO biodistribution of (A) intact chl4.lB antibody and (B) the chl4.18 ~CH2 antibody contruct over time :
within athymic nude mice bearing M21 xenographic .
tumors;
lS FIG. 7 is a graphic representation of the :~
biodistribution of F(ab')2 fragments and the ~CH2 :
antibody construct within nude mice bearing M21 .
~enographic tumors; and FIG. 8 is a graphic representation of the . .
clearance of the AC~2 antibody construct, intact antibody, and F(ab')2 fragments from the circulation of athymic nude mice bearing M21 senographic tumors.
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Descri~tion of the Invention The present invention features truncated antibody constructs having specificity for a 5 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. These ~CH2 constructs are well suited as tarqeting agents 10 for tumor imaging and systemic pharmacotherapeutic treatment.
The invention provides antibody constructs with binding specificity for an epitope on a human 15 tumor antigen. The specificity of the antibody preferably is for a tumor antigen which enables the selective targeting of that ~umor, i.e., the `
construct binds to a unigue marker for the tumor.
Unfortunately, only a few antigen specific markers 20 or human cancers have been identified. However, human tumor cells do have tumor-associated antigens which are present in smaller quantities in certain normal cells such as embryonic cells. Such antigens include alpha fetoprotein (AFP) and carcinoembryonic 25 antigen (CEA). Antibody constructs which recognize these or other tumor-associated antigens such as mucin (B72.3 antibody) and the disialoganglioside GD2 (14.18 antibody) are included in this inven'ion.
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.a., Neuberger et al. (1984) Nature 312:604;
Morrison et al. (1987) Ann. N.Y. Acd. Sci. 507:187; -;, , , - - - - . , , ~ ; . . , . :
~'091/13t66 PCT/~'S91/00633 ~ ~
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.:' '". ', Morrison et al. (1988) Clin. Chem. 34:1668; Horwitz -et al. (1988) Proc. Natl. Acad. Sci. 85:8678). -. . . .
In order to reduce immunogenicity of the 5 ~CH2 constructs in humans, the ~CH2 constructs were designed to ha~e 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 10 regions.
:.
The production of recombinant chimeric antibodies with predetermined specificity has typically involved the use o cloned genomic DNA
15 fragments. For example, the 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). As such, these genomic sequences 20 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 25 acceptor signals at the 5' end of the Ig regions of other species. That is, the splice product between the two maintains the correct reading frame. For example, when a murine V and a human Ck segment are joined and transfected into the appropriate host cell -30 type, the primary transcript is correctly spliced and results in a mature messenger RNA (mRNA) molecule with an open reading frame through both the V and C -~
regions. ~i -:
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..091/13166 PCT/US91/00633 2~773;18 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 5 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 lO chain. This method is described in co-pending patent application serial number 409,889 entitiled "Method of Producing Fusion Proteins", filed September 20, l9~9, herein incorporated as reference.
Briefly, 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 seguence or -eson encoding a variable ~V) region. The cassette is transfected with expressable DNA (structural ge~e) 20 for a constant ~C) region gene having a compatible splice acceptor site at its 5' end.
During the sequence of events leading up to expression in the transected cell of the fusion 25 protein, mRNA derived from the two DNA sequences are spliced to produce a mature mRNA having a 5' end encoding a complete VH or VL and a 3' end encoding a human CH or CL domain. The resulting single chain ~
polypeptide is a fusion of the v region and the -30 constant domain encoded by the exon of the structural gene. In addition, a tumori~idal agent-encoding cassette may be spliced to an Ig C region which, in turn is spliced to an immunoglobulin V region, .~ , ~O91/13166 PCT/US91/00633 resulting in a ~'magic bullet"-type pharmacotherapeutic ~;~
agent.
. .
The cDNA encodes a H or L chain V region 5 (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 (CHl, CH2, and CH3 domains) of another (most likely human) Ig species. Useful V
regions include the VH and VL domains of murine 10 monoclonal antibody 14.18 ~Mujoo et al. (1987) Cancer Res. 47: 1098-1104) which recognizes the disialoganglioside GD2 on the surface of many neuroblastoma, melanoma, glioma, and small lung carcinoma lines and tissues. Other useful V regions 15 include the VH and VL domains of murine monoclonal antibody B72.3 (Johnson et al. (1986) Cancer Res.
46:850) 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 20 the Ig human gamma (H~ and kappa (L) chains.
Alternatively, the V reqion may be of human origin.
Espression of H and L constructs in a single competent host cell results in production of intact chimeric immunoglobulins having a desired specificity 25 and, for example, an intact human constant region.
To produce the ~CH2 construct, a V region cassette is constructed and placed in an appropriate vector, together with a C region-encoding DNA
30 sequence. This C region specifically lacks the CH2 domain and is designed as shown in FIG. 1. FIG. lA
shows a map of the HindIII to PvuII fragment containing the human genomic Cyl gene segment. FIG.
lB shows the DNA encoding the C region of the ~CH2 .
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construct after restriction and ligation of the two fragments with a synthetic linker (to join the Af lIII ~ .
and AvaII ends).
Vectors encoding this mutant C region are transfected into an appropriate host capable of espressing the vectors. Upon co-expression of the two DNAs, the host cell nuclear enzymes produce mRNA
by implementing the normal splicing events, resulting 10 in an mRNA encoding a fused protein. In the case of a chimeric binding protein, the mRNA may encode (5' to 3') a VH or VL domain, which may be identical to the native sequence up to the VC junction, attached directly to another polypeptide such as at least a 15 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 20 intron, resulting in an mRNA encoding the properly fused protein.
:
Normally both exons are placed on the same vector under control of a single regulating -~
25 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.
Particular vector construction, host cell ~ ~ -selection, transformation, and methods of expression do not, per se, constitute an aspect of the --invention, but can be selected and implemented by -, ..
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~'091/13166 PCT/US91/00633 -15- 207~3`~ -skilled workers based on personal preference and convenience. Techniques adaptable for use in the invention are disclosed, for example, in Current Protocols in Molecular Bioloqy (Greene Publishing 5 Associates, 430 Fourth Street, ~rooklyn, 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 lO additional signals or polyadenylation and splicing must be present in cases where they are not provided by the gene itself. For e~ample, all of the signals for the expression of functionally rearranged Ig genes are present on a continuous stretch of DNA and 15 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 ~-20 the signals for e~pression of the selectable phenotype (usually resistance to the lethal effect of a tosic drug such as methotresate, for example).
Therefore, if the vector encodes both the first and second polypeptides, it is necessary that it provide 25 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 30 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. For the .
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production of chimeric antibodies, useful host cells include myelomas or hybridomas such as, for example, the murine non-Ig-producing Sp2~0 Ag 14 hybrido~a cell line, yeast, bacteria, and plant cells. Useful 5 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.
One method for introducing recombinant DNA
10 into cells is electroporation (see, e.g., Potter et al. (1984) Proc. Natl. Acad. Sci. USA 81:7161-7165), which requires specialized equipment and the availability of highly purified DNA. However, many different lines can be transformed using this method 15 if conditions are optimized for the specific cell type.
Another transfection method is protoplast ~spheroplast) fusion ~see, e.g., Sandri-Goldin et al.
20 (1981) ~olec. Cell. Biol. ~:743-752). Bacteria harboring the recombinant plasmid of interest are fused to the lymphoid cells with a chemical agent, generally 45-50% polyethylene glycol i~ a buffered, isotonic solution. This method is simple and does 25 not require extensive purification of plasmid DNA. ~-In addition, 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 30 transfectants containing multiple copies.
Cells which are successfully transformed -~ -with the vector must then be isolated from those which are not. Many methods are available for the ,',~
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~091/13166 PCT/~S91/00633 -17- 20773 1~
selection of transfected cells. For esample, the guanine phosphoribosyl transferase (gpt) and neomycin (neo) resistance markers may be used for selection purposes in lymphoid cells. The gene encoding the 5 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 10 product qenes.
The transfected cell is then cultured to express the polypeptide encoded by the cassette.
Culturing may be in vitro, or in the case of 15 recombinant antibodies, may be accomplished by employing other strategies such as n vivo culturing in ascites fluid.
The CH2 domain of the human Cyl heavy chain 20 which is de~eted from the intact antibody molecule inclùdes the following seguences: the sequence conferring long half-life; the sequence responsible for binding the Fc receptor on e~ector cells; the sequence to which the single N-linked carbohydrate 25 chain is attached; and the sequence to which the first complement component, Clq, binds.
To determine to what extent these sequences are necessary for various antibody functions, the 30 ~C~2 constructs were examined closely. When the supernatants of cells expressing the intact and ~CH2 ~ :
antibodies were first quantitated by measuring associated human ~ chain to normalize the data to the number of antigen binding domains (antibody molecules ~ -. .-. - : . - ., . . .; ;; - .
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contain one ~ 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. ~he chl4.18 ~CH2 5 construct was assayed on GD2-coated plates, while the chB72.3 ~CH2 construct was assayed on mucin-coated plates.
The antigen binding activity of the ~CH2 10 construct was tested further in a competitive binding ;!~ ;
format in order to rule out the possibility o~
non-specific interactions with the ELISA plates. As seen in FIG. 3, the chl4.18 ~CH2 construct competes -much more efficiently with the labeled intact chl4.18 15 antibody for antigen than the normal antibody competes with itself. When the length of the binding assay is estended from 2 hours (FIG. 3A) to 18 hours (~IG. 3B), the difference in binding is not as dramatic, suggesting that the rate of antigen binding 2~ and not the overall affinity is increased by the removal of the CH2 domain.
It is hypothesized that the increased antigen binding of aCH2 antibody constructs reflects 25 conformational changes in the antibody molecule. By removing the CH2 domain, the Fab re~ion (including both V region, CL and CHl sequences) is no longer restricted by interactions with portions of the CH2 domain. Removal of these inter-domain interactions 30 appears to increase the rate of association with antigen. In addition, the removal of the carbohydrate moiety of the CH2 domain may reduce -steric interactions between the antibody and the - antigen.
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20773 !1 g 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.
5 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. Here, the specific lysis o M21 human ~0 melanoma cells in the presence of the indicated concentrations of chl4.18 antibody or ~C~2 construct is shown for an effector to target ratio of lOO~
The average of triplicate samples is shown for each data point. The intact chl4.18 antibody is a very lS potent mediator of ADCC, even at levels as low as l ~
ng/ml. Surprisingly, the ~CH2 construct exhibits a -very small amount of ADCC activity.
The loss of ADCC activity also reflects a 20 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 tc the hinge, it is not surprising that the ~CH2 mutant had greatly reduced activity. For the radioactive 25 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 30 regard.
As expected, the ~CH2 construct also was found to have a reduced ability to mediate the complement lysis of melanoma target cells. FIG. 5 ~. , ... ,, - . ~ .. . .. .. ... . . .
-.~'091/13166 PCT/US91/00633 ~ ~
-20- 2~773~8 ;
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 aCH2 antibodies, having the ability to specifically and rapidly localize to the tumor, also serve as ideal vehicles for the delivery of therapeutic agents. For example, as shown in FIG.
l(C)-l(E), the DNA sequence encoding human lO interleu~in-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 15 T-cells at the tumor site, thereby hastening its destruction. Although the half-life of aCH2 ~ -antibodiès is shorter than whole antibodies, the -~CH2/IL2 fusion protein should be much longer than the half-life of IL~.
~0 . . ':
In another example, the anti-tumor cytokine lymphotoxin or tumor necrosis factor B ~TNF B), could be used to the carboxy terminus of a ~CH2 antibody.
This fusion protein also serves to specifically --25 localize this cytotoxic.protein to a tumor, thus reducing its potential adverse systemic effects.
Likewise, other toxic proteins could be fused to ~CH2 antibodies. These include protein toxins such as ;
ricin, diphtheria toxin, and Pseudomona exotoxin or 30 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.
~091/13166 PCT/US91/00633 -21- 207~3~8 ~
The specificity and half-life of various antibody constructs in vivo can be characterized by performing biodistribution studies. Intact, ~CH2, or F(ab')2 are injected into M21 xenographic tumor- -5 bearing animals, and the amount of construct in various body organs and tissues are monitored over time or at a particular time. FIG. 6 and TABLE 1 ~. .-show the biodistribution of (A) intact chl4.18 antibody and ~B) a ~CH2 construct with time, and lO demonstrates that the latter targets quickly (within 4 hours) and specifically to the tumor.
Time Antibody SamDle 4 hr. 24 hr. 96 hr.
~A) chl4.18 tumor/blood 0.18 0.56 l.08 liver/blood 0.79 2.02 4.55 20 t~) ~CH2 tumor/blood 0.86 2.20 l.l9 liver/blood 2.98 7.37 2.28 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 aCH2 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 ~
', : .
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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.
Because 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 10 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. For ;
example, EGF is fused to an antibody that binds to -15 and activates a cytoto~ic T-cell, thus cross-linkir.g 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 20 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. For example, the vectors constructed to encode the ~CH2 constructs may further encode the tumoricidal 25 protein.
.:
The increased binding activity of the ~CH2 construct, together with its shorter half-life ln v vo, and its low level of nonspecific binding make 30 this molecule extremely useful in the radioactive imaging and/or treatment of tumors. The construc's :
may be labeled with, for example, radionuclides of low energy such as iodine-123, indium-lll, or technetium-9gm, or with any radioactive isotopes that~- ;
. .
' ~ . .
: . -,,, . , . ,-: . ... . .
- ~'0 91/13166 PCl /US91/00633 -23- 20773~ -will not change the binding characteristics of the construct when attached thereto, such as iodine-125.
When labelled with a suitable radioisotope the ~CH2 antibody constructs enable detection of the locus of 5 a tumor-related antigen by external detection of emitted radiation. Radioactive gamma emitters are preerred, although in some circumstances positron emitters, such as 64CU, 11C, and 150 may be used. It is also contemplated that the QC~2 antibody 10 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.
lS 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 20 of the antibody const~uct 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 30 construct, covalently linked on either its 3' or 5' end to a lysine rich polypeptide sequence comprising, for e~ample, 2-20 residues, to serve as a site of attachment for the remotely detectable moieties ., ..... ,. . . , - . .. - . , . . ~ ., . ~ .- . .: . .
. . ,.... .- . .... . . -. . ~ . : :: . ~ - . - . - -.- . . .. . - . - : , . . . . .. . . ..
.~091/13]66 pcT/us91/no633 2 0 7 7 3 ~
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. -For example, diethylenetriamine penta acetic :;
acid (DTPA) may be used to label with Tc-99 or In-lll. In or I isotopes may be attached using the oxine salt (8-hydro~y quinoline). Iodogen (l, 3, 4, "'"!, 10 6 tetrachloro - 3, 6a diphenylglucoluril - Pierce -Chem. Co., Roskville, Ill) may be used for labeling with 125I. (~, e.g., Thakur et al. Thromb. Res.
9:345 (1976); McFarlane, J. Clin. Invest. 42:346 (1963); Knight et al., Radiopharmaceuticals, N.Y.
15 SoC. of Nuclear Medicine, 1975, pp. 149; and Harwig et al., Int. J. Appl. Radiat. Isot. 27:5, 1976.) Other methods are known to those skilled in the art.
ln view of the foregoing, it will be 20 apparent that other types of known reagents can be linked using conventional techniques to ~CH2 antibody contructs. Examples inclùde therapeutic drugs which promote healings such as calcitonin, epidermal growth factor, tumor growth factor alpha and beta, platelet 25 derived growth factor, insulin-like growth factor l (Somatomedin-C), connective tissue activating peptide, and human collagenase inhibitor.
The dosage and means of administration of 30 the family of ~C~2 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 . .
. ' ' . ' ' . ' ~ .'.'. ' ~ . , '. . ' ! ' ~ U'O9l/13166 PCT/~'S91/00633 ... .
-25- 207 ~ 3 `~8 patients, and the nature of the diseased condition in question. When the polypeptide is linked to an imaging agent, it should be administered in an amount sufficient to be detected by scintillation scanning ' ,~
5 or other external radiation detection means capable of detectin~ the localized radiation present in the tumor, such as autoradiography. In general, about 5-20 microcuries should be administered; most preferably about 10 microcuries. The actual amount lO 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 trea,ted by systemic administration of the ~CH2 construct via intravenous injection. Their preferred dosage is in the range of ' from about 0.~1 to lO mg per kg body weight. ~ ' The invention will be further understood ,' from the following, non-limiting esamples.
EXAMPLES
' l. Plasmid Construction ', .
The human Cyl gene, subcloned in pBR322 as a HindIII to PvuII fragment (Gillies et al. (1989) J.
30 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 HindIII to AflIII fragment (containing the CHl and hinge exons) to an AvaII to PvuII
. ~
'O91/13~66 PCT/US91/00633 --.. -. :
-26-20773~g '' '".-"
fragment (containing the CH3 exon) using a synthetic double-stranded oligonucleotide fragment. The ACH2 construct was checked by DNA sequence analysis and then introduced into the chimeric antibody expression 5 vector pdHL2-VCylC~ (Gillies et al. i~id.) 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 10 by first introducing a SmaI site by mutation near the carboxy terminus of the ~CH2 human IgGl gene (FIG.
l(C). The mutation changes nucleotide 2373 (according to the numbering in the Huck et al., (1986) Nucl. Acids. Res. 14:1779-178g) from a T to a 15 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 SmaI site (FIG. lD-lE). The sequence includes, from 5' to 3', the SmaI site, the 2b remaining seguence of the human gamma 1 gene (three , additional amino acids) Sused directly to the first amino acid ~alanine) of the mature I~2 protein sequence, and the amino terminal sequence o IL2 up to the unique PvuII site. The remaining IL2 sequence 2S was then joined at the PvuII site and extends through the coding sequence to a unique XhoI site located 3' -' of the translational stop signal. The XhoI end of the joined DNA was ligatéd to a vector containing the 3' untranslated region and polyA addition site of the 30 mouse ~ constant region (FIG. lF). The resulting construct encodes a ~CH2 human gamma 1 chain to which IL2 is attached at the carboxy terminus.
, ;
.,, ~, ' ~ ' ' ' ' 1 . '; . ' ' ': ' .', , . . : ` ' ~ U'09t/13166 PCT/US91/00633 -27- 20773~8 Transfection and drug selection were carried out essentially as described in Gillies et al.
(Bio/Technol. (1989) 7:799-804), herein incorporated as reference. Briefly, the non-Ig-producing murine 5 hybridoma line, Sp2/0 Agl4, was maintained in Dulbecco~s modified Eagle~s medium (DMEM) containing 10% fetal bovine serum. Plasmids were introduced into cells by protoplast 'fusion essentially as deiscribed in Gillies et al., (1983) Cell 33:
lO 717-728). Transfectants were selected in growth medium containing methotrexate at an initial concentration of O.l ~M. MTX-resistant clones were tested for the secretion of human antibody by ELISA '~' assay. Microtiter plates were coated with goat 15 anti-human IgG (H and L specific-Jackson Laboratories) and incubated with conditioned medium ~' from tran~fected cells. Goat anti-human IgG
~Fc-specific-Jackson Laboratories), conjugated to ' horseradish perosidase, was used for detection of 20 human antibody. The clones that produced the ' greatest amount of antibody were cultured in medium containing increasing concentrations of MTX (from O.l ~M to 1 ~M and after adaptation, to 5 ~ and finally 10 ~M MTX). After a few passages in lO ~M MTX, cells 25 were subcloned by limiting dilution.
3. Purification of Proteins The chl4.18 intact antibodies were purified ~-30 by binding to and elution from protein A Sepharose -(Repligen~. The ~CH2 mutant antibody was purified by immunoaffinity chromatography on an anti-human ~ -monoclonal antibody-Sepharose column. Both proteins were >90~ pure when analyzed by SDS-PAGE or HPLC.
`-~Uosl/l3l66 PCT/US9l/00633 2a7~3ls 4. Antigen Binding Assays Direct antigen binding assays as well as competitive binding assays were performed with the 5 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.). In both cases, the secondary (detecting) antibody was a goat anti-human IgG, 10 Fc-specific polyclonal antiserum (Jackson ImmunoResearch), labeled with horse radish peroxidase (HRP). ~riefly, the GD2-coated plates were first blocked with 5~ bovine serum albumin (BSA) and 5%
goat serum in P8S for 2 hr at 37C. Unlabelled goat ~
15 antibodies were diluted in assay buffer (1% BSA, 1% 7 , .' goat serum in PBS) and 25 ~1 was added to each well.
ASter inculbation at 37C for 4 to 5 hours, 25 ~1 of -HRP-conjugated 14.18 chimeric antibody (6.3 ng) was added to each well. Plates were covereed and -20 incubated overnight at 4C, washed sis times with PBS, and developed with 0-phenylenediamine (OPD) substrate.
5. Cytoto~icity Assays ~ `
ADCC assays were carried out using 5lCr-labeled M21 human melanoma target cells or any cell line which e~presses GD2. A fixed number of labeled targets (2 x 105 cells/ml in 50 ~L) and varying 30 concentrations of human effector cells (peripheral blood leukocytes from normal donors) in 50 ~L were mi~ed with 100 ~L of diluted chimeric antibodies in round-bottom microtiter plates. Following a 4 hr incubation at 37C, the plates were centrifuged, and "
, . : . .
~091~13166 PCT/US91/~0633 ,. . .
20773~8 lO0 ~L samples were removed for counting in a LKB
model 1272 gamma counter.
Complement-mediated lysis of labeled M21 5 melanoma cells was carried out by mixing 50 ~L of cells (2 ~ lOs cells/mL) with an e~ual volume of each antibody dilution. After a 15 min incubation at 37C, lO0 ~L of human complement (1:4 dilution of fresh huma~ serum) was added to each well. Plates lO were incubated for an additional hour at 37C, and the amount of released s~Cr was determined by centrifuging the plates and counting lO0 ~L aliquots of the supernatants. The percentage of cytoto~icity was determined as:
: ', Experimental cpm - Spontaneous cpm ~
Total cpm - Spontaneous cpm ,, .
20 6. Biodistribution and ~lood Clearance Studies , :
8 to lO weeks old female athymic mice ~nu/nu) ~the National Cancer Institute, ~ethesda, MD) were injected subcutaneously with 2 ~ lO6 M21 tumor 25 cells. Tumors of 50-150 mg weight grew within lO
days. At this time, the animals received i.v.
injections into the lateral tail vein of 2S ~g and -3-4 ~Ci l25I-labeled antibody. At designated time points after injection, groups of 3 animals were 30 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, .:
. . , -; .
, ' .. , ' ' '~ , . . ' .,, ~ , . . ,. . ' . ' . ~ . ' ' , . . .
~-~'091/13166 PCT/~IS91/00633 .
skin, muscle, bone, lung, liver, spleen, thyroid, kidney, and intestine) were removed and weighed. All tissue samples were assayed in a gamma counter for l25I activity. The results were calculated as -5 percent of injected dose per g tissue and as -localization ratios (cpm/g tumor : cpm/g tissue).
.. . .
2~77348 - -For example, several days after the administration of radiolabelled, intact antibody IgG, the level of circulating and nonspecifically bound radioactivity is high, and clearance occurs gradually over a period 5 of four days or more (Goldenberg, ibid.). In -comparison, 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 123I and 99mTc (Goldenberg, 10 ibid.).
However, the use of F(ab')2 fragments is not without pitfalls. Besides being difficult to produce enzymatically, the binding properties of these 15 fragments may be compromised (~ç~, e.a., Wahl et al., ibid. Therefore, what is needed is a targeting molecule ~aving specificity, enhanced binding activity, minimal nonspecific binding abilities, and a shorter half-life in vivo.
Towards this end, monoclonal antibodies with increased antigen binding activites have been identified which have mUtationS in their constant regions ~see, e.g., Pollock et al. (1988) Proc. Natl.
25 Acad. Sci. USA 85:2298-2302). In addition, the ability to genetically engineer and re-express -antibodies in transfected cells has made it possible to combine various portions of the immunoglobulin molecule as fusion proteins (Neuberger et al. (1984) , 30 Nature 312:614; Morrison et al. (1987~ Ann. N.Y.
Acad. Sci. 507:187; and Morrison et al. (1988) Clin. ;
Chem. 34:1668). It is also possible to genetically - encode immunoglobulin deletion mutants such as Fab (Horwitz et al. (1988) Proc. Natl. Acad. Sci. U.S.A. -",~
' .O9l/13166 PCT/US91/00633 2077~18 85:8687) or F(ab')2-like fragments. In one study, an Fd~ fragment, a truncated form of the immunoglobulin heavy chain, was expressed in E. coli (Cabilly et al.
(1984) Proc. Natl. Acad. Sci. USA 81:3273-3277). In 5 another study, specific constant region domains of the human Cy3 H chain were either deleted or added in order to assess the effect on antibody synthesis and assembly (Morrison et al. (1987) Ann. N.Y. Acad. Sci.
507:1873).
Accordingly, it is 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 15 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 oS the body is reduced. Additional 20 ob~ects 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.
...... ........ . . . . ~ . . -, . . - -.. ,. .- .~. - ~ - . - . . -, . . . . . .. .
, . - ... ,.. - ,. . -. , ~: . : ., .:~ . ............. .. . . .
: , .:,. . . - - . -: ~ , ... . , , .. . . :
- ~O9ltl3166 PCT/US91/00633 20773~8 ... ... . . . .
summary of the Invention It has been discovered that antibody constructs retaining their CHl and CH3 domains, but 5 lacking their CH2 domain, surprisingly have an enhanced binding affinity as well as a shorter half-life and an unexpectedly low level of non-specific binding relative to an intact antibody molécule with the same specificity. This finding has 10 been exploited to develop the present invention which features antibody constructs having specificity for an epitope, present on various tumor antigens.
Native antibody molecules of the IgG and IgD
15 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 (~) domains 2t the amino termini of the two chains together form the antigen binding region. The 20 first constant ~C) domain of the H chain (CH1) interacts with the C region of the L chain through hydrophobic interactions and also through a disulfide bond. The ne~t H-chàin domain, adjacent to the hinge, is called CH2. This domain reportedly 25 contains many o the effector functions of the antibody including the sequences responsible or complement fixation and Fc receptor-mediated -antibody-dependent cellular cytotoxicity (ADCC), and is the sole N-linked glycosylation site in human Cyl 30 chains. The CH3 domain at the carboxy terminus of the H-chain is thought to play a major role in the assembly of H chains. :
-, ~`. ' ' `'.,, ', ' "`" ' . '~` ;~' .,,'.' '.' .
:_0 91tl3166 PCI/US91/00633 2077~48 The antibody constructs of the present invention include a first portion having an immunoglobulin binding region of predetermined specificity. This binding region has plural 5 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 CHl domain and a hinge whose C terminus is linked to the N
10 terminus of a CH3 domain, i.e., a hybrid constant domain which omits the CH2 domain normally present in a native antibody. ~.ntibody constructs with the CH2 domain deleted are referred to hereinafter as ~CH2 constructs. ~CH2 constructs are characterized by 15 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.
. .
In one preferred embodiment, the binding region of the ~CH2 antibody construct includes the binding region of a nonhuman immunoglobulin such as a murine immunoglobulin. In one aspect o~ the invention, the immunoglobulin binding region is-one 25 which has a predetermined specificity for mucin such - -as the binding region of monoclonal antibody B72.3, or has a predetermined specificity for the GD2 - -ganglioside such as the binding region of monoclonal antibody 14.18. In another embodiment of the 30 invention, the immunoglobulin binding region has a predetermined specificity for a melanoma-specific proteoglycan. The preferred altered constant region - -- includes a human immunoglobulin constant domain.
. . . . . . . . . . . . ~ . . . . . . . , . . . . , . . . . . . . . = . . ~ - . . . . . .
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,~'091/13166 PCT/US91/0063~ ~
_7_ 2Q77~`~18 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 carbo~y terminus of the the second portion comprising a tumoricidal protein.
This third protein portion preferably is lymphotoxin, interleukin-2, epidermal growth factor ~EGF), active lO analogs thereof, or active fragments thereof. In a preferred embodiment of the invention, the third portion is peptide bonded via an endopeptidase-cleavable amino acid residue, such as lysine, to the carbo~y terminus of the CH3- domain of the second 15 region. Alternatively, the third portion may be linked to the second portion by chemical crosslinking, for esample.
... . ..
In yet another embodiment, for use as an 20 imaging agent, the ~CH2 antibody construct includes a radioactive label attached thereto.
~-'-.
:
~.091tl3166 PCI/I,IS91/00633 20773~8 Brief Description of the Drawing The foregoing and other objects of the -present invention, the various features thereof, as 5 well as the inventions thereof may be more ~ully ~ ~
understood from the following description when read ~ --together with the accompanying drawings in which:
FIG. 1 is a schematic representation showing 10 the construction of a ~CH2 deletion mutant form of , the hu~an Cyl gene: (A) is a map of the HindIII to PvuII fragment containing the genomic Cyl gene segment and restriction sites; (B) is the ~CH2 deleted gene missing the AflIII to AvaII fragment 15 (the CH2 exon) after restriction and ligation; (C) shows the SmaI site engineered in the CH3 gene; (D) is a map of a SmaI to PvuII linker which is used to ~
at~ach the (E) PvuII to XhoI fragment containing an :
I~2 gene; and ~F) is is the remainder of the vector 20 including an XhoI site and polyA region;
, FIG. 2 is a graphic representation of the antigen binding activity of variaus ~C~2 chimeric constructs: (A) ~he ~CH2 chl4.18 antibody construct;
25 (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 30 chl4.12 antibody and the ~CH2 chl4.18 antibody constructs after (A) 2 hours incubation at 37C; and (B) 18 hours incubation at 4C;
.
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. .~'091/13166 PCT/~'S91/00633 2 ~ 7 7 ~
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 lO biodistribution of (A) intact chl4.lB antibody and (B) the chl4.18 ~CH2 antibody contruct over time :
within athymic nude mice bearing M21 xenographic .
tumors;
lS FIG. 7 is a graphic representation of the :~
biodistribution of F(ab')2 fragments and the ~CH2 :
antibody construct within nude mice bearing M21 .
~enographic tumors; and FIG. 8 is a graphic representation of the . .
clearance of the AC~2 antibody construct, intact antibody, and F(ab')2 fragments from the circulation of athymic nude mice bearing M21 senographic tumors.
. .
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,O91/13166 PCT/US91/0063~
-lO- 2077 3~18 -:
Descri~tion of the Invention The present invention features truncated antibody constructs having specificity for a 5 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. These ~CH2 constructs are well suited as tarqeting agents 10 for tumor imaging and systemic pharmacotherapeutic treatment.
The invention provides antibody constructs with binding specificity for an epitope on a human 15 tumor antigen. The specificity of the antibody preferably is for a tumor antigen which enables the selective targeting of that ~umor, i.e., the `
construct binds to a unigue marker for the tumor.
Unfortunately, only a few antigen specific markers 20 or human cancers have been identified. However, human tumor cells do have tumor-associated antigens which are present in smaller quantities in certain normal cells such as embryonic cells. Such antigens include alpha fetoprotein (AFP) and carcinoembryonic 25 antigen (CEA). Antibody constructs which recognize these or other tumor-associated antigens such as mucin (B72.3 antibody) and the disialoganglioside GD2 (14.18 antibody) are included in this inven'ion.
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.a., Neuberger et al. (1984) Nature 312:604;
Morrison et al. (1987) Ann. N.Y. Acd. Sci. 507:187; -;, , , - - - - . , , ~ ; . . , . :
~'091/13t66 PCT/~'S91/00633 ~ ~
-11- 20773'~8 ::
.:' '". ', Morrison et al. (1988) Clin. Chem. 34:1668; Horwitz -et al. (1988) Proc. Natl. Acad. Sci. 85:8678). -. . . .
In order to reduce immunogenicity of the 5 ~CH2 constructs in humans, the ~CH2 constructs were designed to ha~e 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 10 regions.
:.
The production of recombinant chimeric antibodies with predetermined specificity has typically involved the use o cloned genomic DNA
15 fragments. For example, the 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). As such, these genomic sequences 20 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 25 acceptor signals at the 5' end of the Ig regions of other species. That is, the splice product between the two maintains the correct reading frame. For example, when a murine V and a human Ck segment are joined and transfected into the appropriate host cell -30 type, the primary transcript is correctly spliced and results in a mature messenger RNA (mRNA) molecule with an open reading frame through both the V and C -~
regions. ~i -:
~ ' - . .
..091/13166 PCT/US91/00633 2~773;18 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 5 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 lO chain. This method is described in co-pending patent application serial number 409,889 entitiled "Method of Producing Fusion Proteins", filed September 20, l9~9, herein incorporated as reference.
Briefly, 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 seguence or -eson encoding a variable ~V) region. The cassette is transfected with expressable DNA (structural ge~e) 20 for a constant ~C) region gene having a compatible splice acceptor site at its 5' end.
During the sequence of events leading up to expression in the transected cell of the fusion 25 protein, mRNA derived from the two DNA sequences are spliced to produce a mature mRNA having a 5' end encoding a complete VH or VL and a 3' end encoding a human CH or CL domain. The resulting single chain ~
polypeptide is a fusion of the v region and the -30 constant domain encoded by the exon of the structural gene. In addition, a tumori~idal agent-encoding cassette may be spliced to an Ig C region which, in turn is spliced to an immunoglobulin V region, .~ , ~O91/13166 PCT/US91/00633 resulting in a ~'magic bullet"-type pharmacotherapeutic ~;~
agent.
. .
The cDNA encodes a H or L chain V region 5 (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 (CHl, CH2, and CH3 domains) of another (most likely human) Ig species. Useful V
regions include the VH and VL domains of murine 10 monoclonal antibody 14.18 ~Mujoo et al. (1987) Cancer Res. 47: 1098-1104) which recognizes the disialoganglioside GD2 on the surface of many neuroblastoma, melanoma, glioma, and small lung carcinoma lines and tissues. Other useful V regions 15 include the VH and VL domains of murine monoclonal antibody B72.3 (Johnson et al. (1986) Cancer Res.
46:850) 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 20 the Ig human gamma (H~ and kappa (L) chains.
Alternatively, the V reqion may be of human origin.
Espression of H and L constructs in a single competent host cell results in production of intact chimeric immunoglobulins having a desired specificity 25 and, for example, an intact human constant region.
To produce the ~CH2 construct, a V region cassette is constructed and placed in an appropriate vector, together with a C region-encoding DNA
30 sequence. This C region specifically lacks the CH2 domain and is designed as shown in FIG. 1. FIG. lA
shows a map of the HindIII to PvuII fragment containing the human genomic Cyl gene segment. FIG.
lB shows the DNA encoding the C region of the ~CH2 .
, , - ` : : .
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-..091/13166 PCT/US91/00633 ' . :
20773~8 ~ : :
construct after restriction and ligation of the two fragments with a synthetic linker (to join the Af lIII ~ .
and AvaII ends).
Vectors encoding this mutant C region are transfected into an appropriate host capable of espressing the vectors. Upon co-expression of the two DNAs, the host cell nuclear enzymes produce mRNA
by implementing the normal splicing events, resulting 10 in an mRNA encoding a fused protein. In the case of a chimeric binding protein, the mRNA may encode (5' to 3') a VH or VL domain, which may be identical to the native sequence up to the VC junction, attached directly to another polypeptide such as at least a 15 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 20 intron, resulting in an mRNA encoding the properly fused protein.
:
Normally both exons are placed on the same vector under control of a single regulating -~
25 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.
Particular vector construction, host cell ~ ~ -selection, transformation, and methods of expression do not, per se, constitute an aspect of the --invention, but can be selected and implemented by -, ..
..
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~'091/13166 PCT/US91/00633 -15- 207~3`~ -skilled workers based on personal preference and convenience. Techniques adaptable for use in the invention are disclosed, for example, in Current Protocols in Molecular Bioloqy (Greene Publishing 5 Associates, 430 Fourth Street, ~rooklyn, 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 lO additional signals or polyadenylation and splicing must be present in cases where they are not provided by the gene itself. For e~ample, all of the signals for the expression of functionally rearranged Ig genes are present on a continuous stretch of DNA and 15 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 ~-20 the signals for e~pression of the selectable phenotype (usually resistance to the lethal effect of a tosic drug such as methotresate, for example).
Therefore, if the vector encodes both the first and second polypeptides, it is necessary that it provide 25 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 30 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. For the .
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-,iO 91/13166 P ~ /US91/00633 2~773~
production of chimeric antibodies, useful host cells include myelomas or hybridomas such as, for example, the murine non-Ig-producing Sp2~0 Ag 14 hybrido~a cell line, yeast, bacteria, and plant cells. Useful 5 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.
One method for introducing recombinant DNA
10 into cells is electroporation (see, e.g., Potter et al. (1984) Proc. Natl. Acad. Sci. USA 81:7161-7165), which requires specialized equipment and the availability of highly purified DNA. However, many different lines can be transformed using this method 15 if conditions are optimized for the specific cell type.
Another transfection method is protoplast ~spheroplast) fusion ~see, e.g., Sandri-Goldin et al.
20 (1981) ~olec. Cell. Biol. ~:743-752). Bacteria harboring the recombinant plasmid of interest are fused to the lymphoid cells with a chemical agent, generally 45-50% polyethylene glycol i~ a buffered, isotonic solution. This method is simple and does 25 not require extensive purification of plasmid DNA. ~-In addition, 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 30 transfectants containing multiple copies.
Cells which are successfully transformed -~ -with the vector must then be isolated from those which are not. Many methods are available for the ,',~
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~091/13166 PCT/~S91/00633 -17- 20773 1~
selection of transfected cells. For esample, the guanine phosphoribosyl transferase (gpt) and neomycin (neo) resistance markers may be used for selection purposes in lymphoid cells. The gene encoding the 5 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 10 product qenes.
The transfected cell is then cultured to express the polypeptide encoded by the cassette.
Culturing may be in vitro, or in the case of 15 recombinant antibodies, may be accomplished by employing other strategies such as n vivo culturing in ascites fluid.
The CH2 domain of the human Cyl heavy chain 20 which is de~eted from the intact antibody molecule inclùdes the following seguences: the sequence conferring long half-life; the sequence responsible for binding the Fc receptor on e~ector cells; the sequence to which the single N-linked carbohydrate 25 chain is attached; and the sequence to which the first complement component, Clq, binds.
To determine to what extent these sequences are necessary for various antibody functions, the 30 ~C~2 constructs were examined closely. When the supernatants of cells expressing the intact and ~CH2 ~ :
antibodies were first quantitated by measuring associated human ~ chain to normalize the data to the number of antigen binding domains (antibody molecules ~ -. .-. - : . - ., . . .; ;; - .
- .. - . .. ; . . ......... . .
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~'0 ~1/13166 PCT/US91/00633 -18- 2~7 73ll~
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contain one ~ 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. ~he chl4.18 ~CH2 5 construct was assayed on GD2-coated plates, while the chB72.3 ~CH2 construct was assayed on mucin-coated plates.
The antigen binding activity of the ~CH2 10 construct was tested further in a competitive binding ;!~ ;
format in order to rule out the possibility o~
non-specific interactions with the ELISA plates. As seen in FIG. 3, the chl4.18 ~CH2 construct competes -much more efficiently with the labeled intact chl4.18 15 antibody for antigen than the normal antibody competes with itself. When the length of the binding assay is estended from 2 hours (FIG. 3A) to 18 hours (~IG. 3B), the difference in binding is not as dramatic, suggesting that the rate of antigen binding 2~ and not the overall affinity is increased by the removal of the CH2 domain.
It is hypothesized that the increased antigen binding of aCH2 antibody constructs reflects 25 conformational changes in the antibody molecule. By removing the CH2 domain, the Fab re~ion (including both V region, CL and CHl sequences) is no longer restricted by interactions with portions of the CH2 domain. Removal of these inter-domain interactions 30 appears to increase the rate of association with antigen. In addition, the removal of the carbohydrate moiety of the CH2 domain may reduce -steric interactions between the antibody and the - antigen.
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~091/13166 PCT/~S91/00633 .
20773 !1 g 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.
5 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. Here, the specific lysis o M21 human ~0 melanoma cells in the presence of the indicated concentrations of chl4.18 antibody or ~C~2 construct is shown for an effector to target ratio of lOO~
The average of triplicate samples is shown for each data point. The intact chl4.18 antibody is a very lS potent mediator of ADCC, even at levels as low as l ~
ng/ml. Surprisingly, the ~CH2 construct exhibits a -very small amount of ADCC activity.
The loss of ADCC activity also reflects a 20 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 tc the hinge, it is not surprising that the ~CH2 mutant had greatly reduced activity. For the radioactive 25 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 30 regard.
As expected, the ~CH2 construct also was found to have a reduced ability to mediate the complement lysis of melanoma target cells. FIG. 5 ~. , ... ,, - . ~ .. . .. .. ... . . .
-.~'091/13166 PCT/US91/00633 ~ ~
-20- 2~773~8 ;
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 aCH2 antibodies, having the ability to specifically and rapidly localize to the tumor, also serve as ideal vehicles for the delivery of therapeutic agents. For example, as shown in FIG.
l(C)-l(E), the DNA sequence encoding human lO interleu~in-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 15 T-cells at the tumor site, thereby hastening its destruction. Although the half-life of aCH2 ~ -antibodiès is shorter than whole antibodies, the -~CH2/IL2 fusion protein should be much longer than the half-life of IL~.
~0 . . ':
In another example, the anti-tumor cytokine lymphotoxin or tumor necrosis factor B ~TNF B), could be used to the carboxy terminus of a ~CH2 antibody.
This fusion protein also serves to specifically --25 localize this cytotoxic.protein to a tumor, thus reducing its potential adverse systemic effects.
Likewise, other toxic proteins could be fused to ~CH2 antibodies. These include protein toxins such as ;
ricin, diphtheria toxin, and Pseudomona exotoxin or 30 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.
~091/13166 PCT/US91/00633 -21- 207~3~8 ~
The specificity and half-life of various antibody constructs in vivo can be characterized by performing biodistribution studies. Intact, ~CH2, or F(ab')2 are injected into M21 xenographic tumor- -5 bearing animals, and the amount of construct in various body organs and tissues are monitored over time or at a particular time. FIG. 6 and TABLE 1 ~. .-show the biodistribution of (A) intact chl4.18 antibody and ~B) a ~CH2 construct with time, and lO demonstrates that the latter targets quickly (within 4 hours) and specifically to the tumor.
Time Antibody SamDle 4 hr. 24 hr. 96 hr.
~A) chl4.18 tumor/blood 0.18 0.56 l.08 liver/blood 0.79 2.02 4.55 20 t~) ~CH2 tumor/blood 0.86 2.20 l.l9 liver/blood 2.98 7.37 2.28 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 aCH2 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 ~
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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.
Because 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 10 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. For ;
example, EGF is fused to an antibody that binds to -15 and activates a cytoto~ic T-cell, thus cross-linkir.g 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 20 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. For example, the vectors constructed to encode the ~CH2 constructs may further encode the tumoricidal 25 protein.
.:
The increased binding activity of the ~CH2 construct, together with its shorter half-life ln v vo, and its low level of nonspecific binding make 30 this molecule extremely useful in the radioactive imaging and/or treatment of tumors. The construc's :
may be labeled with, for example, radionuclides of low energy such as iodine-123, indium-lll, or technetium-9gm, or with any radioactive isotopes that~- ;
. .
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- ~'0 91/13166 PCl /US91/00633 -23- 20773~ -will not change the binding characteristics of the construct when attached thereto, such as iodine-125.
When labelled with a suitable radioisotope the ~CH2 antibody constructs enable detection of the locus of 5 a tumor-related antigen by external detection of emitted radiation. Radioactive gamma emitters are preerred, although in some circumstances positron emitters, such as 64CU, 11C, and 150 may be used. It is also contemplated that the QC~2 antibody 10 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.
lS 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 20 of the antibody const~uct 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 30 construct, covalently linked on either its 3' or 5' end to a lysine rich polypeptide sequence comprising, for e~ample, 2-20 residues, to serve as a site of attachment for the remotely detectable moieties ., ..... ,. . . , - . .. - . , . . ~ ., . ~ .- . .: . .
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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. -For example, diethylenetriamine penta acetic :;
acid (DTPA) may be used to label with Tc-99 or In-lll. In or I isotopes may be attached using the oxine salt (8-hydro~y quinoline). Iodogen (l, 3, 4, "'"!, 10 6 tetrachloro - 3, 6a diphenylglucoluril - Pierce -Chem. Co., Roskville, Ill) may be used for labeling with 125I. (~, e.g., Thakur et al. Thromb. Res.
9:345 (1976); McFarlane, J. Clin. Invest. 42:346 (1963); Knight et al., Radiopharmaceuticals, N.Y.
15 SoC. of Nuclear Medicine, 1975, pp. 149; and Harwig et al., Int. J. Appl. Radiat. Isot. 27:5, 1976.) Other methods are known to those skilled in the art.
ln view of the foregoing, it will be 20 apparent that other types of known reagents can be linked using conventional techniques to ~CH2 antibody contructs. Examples inclùde therapeutic drugs which promote healings such as calcitonin, epidermal growth factor, tumor growth factor alpha and beta, platelet 25 derived growth factor, insulin-like growth factor l (Somatomedin-C), connective tissue activating peptide, and human collagenase inhibitor.
The dosage and means of administration of 30 the family of ~C~2 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 . .
. ' ' . ' ' . ' ~ .'.'. ' ~ . , '. . ' ! ' ~ U'O9l/13166 PCT/~'S91/00633 ... .
-25- 207 ~ 3 `~8 patients, and the nature of the diseased condition in question. When the polypeptide is linked to an imaging agent, it should be administered in an amount sufficient to be detected by scintillation scanning ' ,~
5 or other external radiation detection means capable of detectin~ the localized radiation present in the tumor, such as autoradiography. In general, about 5-20 microcuries should be administered; most preferably about 10 microcuries. The actual amount lO 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 trea,ted by systemic administration of the ~CH2 construct via intravenous injection. Their preferred dosage is in the range of ' from about 0.~1 to lO mg per kg body weight. ~ ' The invention will be further understood ,' from the following, non-limiting esamples.
EXAMPLES
' l. Plasmid Construction ', .
The human Cyl gene, subcloned in pBR322 as a HindIII to PvuII fragment (Gillies et al. (1989) J.
30 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 HindIII to AflIII fragment (containing the CHl and hinge exons) to an AvaII to PvuII
. ~
'O91/13~66 PCT/US91/00633 --.. -. :
-26-20773~g '' '".-"
fragment (containing the CH3 exon) using a synthetic double-stranded oligonucleotide fragment. The ACH2 construct was checked by DNA sequence analysis and then introduced into the chimeric antibody expression 5 vector pdHL2-VCylC~ (Gillies et al. i~id.) 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 10 by first introducing a SmaI site by mutation near the carboxy terminus of the ~CH2 human IgGl gene (FIG.
l(C). The mutation changes nucleotide 2373 (according to the numbering in the Huck et al., (1986) Nucl. Acids. Res. 14:1779-178g) from a T to a 15 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 SmaI site (FIG. lD-lE). The sequence includes, from 5' to 3', the SmaI site, the 2b remaining seguence of the human gamma 1 gene (three , additional amino acids) Sused directly to the first amino acid ~alanine) of the mature I~2 protein sequence, and the amino terminal sequence o IL2 up to the unique PvuII site. The remaining IL2 sequence 2S was then joined at the PvuII site and extends through the coding sequence to a unique XhoI site located 3' -' of the translational stop signal. The XhoI end of the joined DNA was ligatéd to a vector containing the 3' untranslated region and polyA addition site of the 30 mouse ~ constant region (FIG. lF). The resulting construct encodes a ~CH2 human gamma 1 chain to which IL2 is attached at the carboxy terminus.
, ;
.,, ~, ' ~ ' ' ' ' 1 . '; . ' ' ': ' .', , . . : ` ' ~ U'09t/13166 PCT/US91/00633 -27- 20773~8 Transfection and drug selection were carried out essentially as described in Gillies et al.
(Bio/Technol. (1989) 7:799-804), herein incorporated as reference. Briefly, the non-Ig-producing murine 5 hybridoma line, Sp2/0 Agl4, was maintained in Dulbecco~s modified Eagle~s medium (DMEM) containing 10% fetal bovine serum. Plasmids were introduced into cells by protoplast 'fusion essentially as deiscribed in Gillies et al., (1983) Cell 33:
lO 717-728). Transfectants were selected in growth medium containing methotrexate at an initial concentration of O.l ~M. MTX-resistant clones were tested for the secretion of human antibody by ELISA '~' assay. Microtiter plates were coated with goat 15 anti-human IgG (H and L specific-Jackson Laboratories) and incubated with conditioned medium ~' from tran~fected cells. Goat anti-human IgG
~Fc-specific-Jackson Laboratories), conjugated to ' horseradish perosidase, was used for detection of 20 human antibody. The clones that produced the ' greatest amount of antibody were cultured in medium containing increasing concentrations of MTX (from O.l ~M to 1 ~M and after adaptation, to 5 ~ and finally 10 ~M MTX). After a few passages in lO ~M MTX, cells 25 were subcloned by limiting dilution.
3. Purification of Proteins The chl4.18 intact antibodies were purified ~-30 by binding to and elution from protein A Sepharose -(Repligen~. The ~CH2 mutant antibody was purified by immunoaffinity chromatography on an anti-human ~ -monoclonal antibody-Sepharose column. Both proteins were >90~ pure when analyzed by SDS-PAGE or HPLC.
`-~Uosl/l3l66 PCT/US9l/00633 2a7~3ls 4. Antigen Binding Assays Direct antigen binding assays as well as competitive binding assays were performed with the 5 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.). In both cases, the secondary (detecting) antibody was a goat anti-human IgG, 10 Fc-specific polyclonal antiserum (Jackson ImmunoResearch), labeled with horse radish peroxidase (HRP). ~riefly, the GD2-coated plates were first blocked with 5~ bovine serum albumin (BSA) and 5%
goat serum in P8S for 2 hr at 37C. Unlabelled goat ~
15 antibodies were diluted in assay buffer (1% BSA, 1% 7 , .' goat serum in PBS) and 25 ~1 was added to each well.
ASter inculbation at 37C for 4 to 5 hours, 25 ~1 of -HRP-conjugated 14.18 chimeric antibody (6.3 ng) was added to each well. Plates were covereed and -20 incubated overnight at 4C, washed sis times with PBS, and developed with 0-phenylenediamine (OPD) substrate.
5. Cytoto~icity Assays ~ `
ADCC assays were carried out using 5lCr-labeled M21 human melanoma target cells or any cell line which e~presses GD2. A fixed number of labeled targets (2 x 105 cells/ml in 50 ~L) and varying 30 concentrations of human effector cells (peripheral blood leukocytes from normal donors) in 50 ~L were mi~ed with 100 ~L of diluted chimeric antibodies in round-bottom microtiter plates. Following a 4 hr incubation at 37C, the plates were centrifuged, and "
, . : . .
~091~13166 PCT/US91/~0633 ,. . .
20773~8 lO0 ~L samples were removed for counting in a LKB
model 1272 gamma counter.
Complement-mediated lysis of labeled M21 5 melanoma cells was carried out by mixing 50 ~L of cells (2 ~ lOs cells/mL) with an e~ual volume of each antibody dilution. After a 15 min incubation at 37C, lO0 ~L of human complement (1:4 dilution of fresh huma~ serum) was added to each well. Plates lO were incubated for an additional hour at 37C, and the amount of released s~Cr was determined by centrifuging the plates and counting lO0 ~L aliquots of the supernatants. The percentage of cytoto~icity was determined as:
: ', Experimental cpm - Spontaneous cpm ~
Total cpm - Spontaneous cpm ,, .
20 6. Biodistribution and ~lood Clearance Studies , :
8 to lO weeks old female athymic mice ~nu/nu) ~the National Cancer Institute, ~ethesda, MD) were injected subcutaneously with 2 ~ lO6 M21 tumor 25 cells. Tumors of 50-150 mg weight grew within lO
days. At this time, the animals received i.v.
injections into the lateral tail vein of 2S ~g and -3-4 ~Ci l25I-labeled antibody. At designated time points after injection, groups of 3 animals were 30 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, .:
. . , -; .
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~-~'091/13166 PCT/~IS91/00633 .
skin, muscle, bone, lung, liver, spleen, thyroid, kidney, and intestine) were removed and weighed. All tissue samples were assayed in a gamma counter for l25I activity. The results were calculated as -5 percent of injected dose per g tissue and as -localization ratios (cpm/g tumor : cpm/g tissue).
.. . .
7. Radioactive Labeling Chl4.18, chl4.lB-~CH2, and F(ab')2 fragments of human IgG were labeled with l25I. Briefly, 500 ~9 antibody was incubated for 25 min on ice with 0.5 mCi l25I (lO0 mCi or 3.75 Gaq/ml, Amersham Corp., Arlington Heights, IL) in polystyrene tubes coated 15 with lO0 ~g Iodo-Gen reagent (Pierce Chemical Co., Rockford, IL). Unincorporated l25I was removed by gel filtration on PDlO columns (Pharmacia Fine ~
Chemicals, Piscataway, NJ). Specific activity was -typically 1.5 - 0.5 nCI/ng antibody.
The invention may be embodied in other specific forms without departing ~rom the spirit or essential characteristics thereof. The present embodiments are therefore considered to be in all 25 respects as illustrative and not restrictive, the scope of the invention beinq indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are 30 therefore intended to be embraced therein.
; ~ What is claimed is:
' : ' ... . , ,~ . - ... i . -, ~ .. , . ;. ., , . -.. - . - . . . : ... . . ~ . , ~
. ~ . .. .. : . - ., . ... , . ~ : :
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Chemicals, Piscataway, NJ). Specific activity was -typically 1.5 - 0.5 nCI/ng antibody.
The invention may be embodied in other specific forms without departing ~rom the spirit or essential characteristics thereof. The present embodiments are therefore considered to be in all 25 respects as illustrative and not restrictive, the scope of the invention beinq indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are 30 therefore intended to be embraced therein.
; ~ What is claimed is:
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Claims (15)
1. An antibody construct specific for an epitope on an antigen, said construct comprising:
(a) an immunoglobulin binding region of predetermined specificity comprising hypervariable regions homologous with a hypervariable region of a native antibody specific for said epitope; and (b) an immunoglobulin constant region consisting essentially of a CH1 domain, a hinge, and a CH3 domain and omitting the CH2 domain normally present in a native antibody, said constant region enhancing the binding acitivity of said antibody construct at least two fold in comparison with the binding activity of said native antibody.
(a) an immunoglobulin binding region of predetermined specificity comprising hypervariable regions homologous with a hypervariable region of a native antibody specific for said epitope; and (b) an immunoglobulin constant region consisting essentially of a CH1 domain, a hinge, and a CH3 domain and omitting the CH2 domain normally present in a native antibody, said constant region enhancing the binding acitivity of said antibody construct at least two fold in comparison with the binding activity of said native antibody.
2. The construct of claim 1 wherein said binding region comprises a binding region of a nonhuman immunoglobulin molecule.
3. The construct of claim 2 wherein said binding region comprises a binding region of a murine immunoglobulin molecule.
4. The construct of claim 1 wherein said binding region has a specificity for mucin.
5. The construct of claim 1 wherein said binding region has a specificity for GD2 ganglioside.
6. The construct of claim 1 wherein said binding region has a specificity for a melanoma-specific proteoglycan.
7. The construct of claim 1 wherein said constant region comprises a human immunoglobulin constant domain.
8. The construct of claim 1 comprising an immunoglobulin heavy chain disulfide-linked to an immunoglobulin light chain.
9. The construct of claim 1 further comprising, attached to the carboxy terminus of said constant region, a protein having tumoricidal activity.
10. The construct of claim 9 wherein said protein is peptide bonded via an endopeptidase-cleavable amino acid sequence to the carboxy terminus of said CH3 domain.
11. The antibody construct of claim 10 wherein said protein is peptide bonded via a lysine residue to the carboxy terminus of said CH3 domain.
12. The construct of claim 9 wherein said protein comprises a tumoricidal protein selected from the group consisting of lymphotoxin, active analogs thereof, and active fragments thereof.
13. The construct of claim 9 wherein said protein comprises a tumoricidal protein selected from the group consisting of interleukin-2, active analogs thereof, and active fragments thereof.
14. The antibody construct of claim 9 wherein said protein comprises a tumoricidal protein selected from the group consisting of epidermal growth factor, tumor necrosis factor, ricin, diptheria toxin, Psuedomona exotoxin, active analogs thereof, and active fragments thereof.
15. The construct of claim 1 further comprising a radioactive label.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US488,297 | 1983-04-25 | ||
| US48829790A | 1990-03-02 | 1990-03-02 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2077348A1 true CA2077348A1 (en) | 1991-09-03 |
Family
ID=23939159
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002077348A Abandoned CA2077348A1 (en) | 1990-03-02 | 1991-01-30 | Antibody constructs with enhanced binding affinity |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0517754A1 (en) |
| JP (1) | JPH07507440A (en) |
| CA (1) | CA2077348A1 (en) |
| WO (1) | WO1991013166A1 (en) |
Families Citing this family (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5650150A (en) * | 1990-11-09 | 1997-07-22 | Gillies; Stephen D. | Recombinant antibody cytokine fusion proteins |
| ATE218889T1 (en) * | 1990-11-09 | 2002-06-15 | Stephen D Gillies | CYTOKINE IMMUNOCONJUGATES |
| EP1042459A4 (en) * | 1997-12-24 | 2003-07-23 | Diatech Pty Ltd | Bifunctional molecules |
| US6617135B1 (en) | 1999-08-09 | 2003-09-09 | Emd Lexigen Research Center Corp. | Multiple cytokine protein complexes |
| US7754208B2 (en) | 2001-01-17 | 2010-07-13 | Trubion Pharmaceuticals, Inc. | Binding domain-immunoglobulin fusion proteins |
| US20030133939A1 (en) | 2001-01-17 | 2003-07-17 | Genecraft, Inc. | Binding domain-immunoglobulin fusion proteins |
| MXPA03008031A (en) * | 2001-03-07 | 2003-12-04 | Merck Patent Gmbh | Expression technology for proteins containing a hybrid isotype antibody moiety. |
| WO2002079415A2 (en) | 2001-03-30 | 2002-10-10 | Lexigen Pharmaceuticals Corp. | Reducing the immunogenicity of fusion proteins |
| CA2534959A1 (en) * | 2003-09-05 | 2005-03-31 | Genentech, Inc. | Antibodies with altered effector functions |
| EP1706428B1 (en) | 2004-01-22 | 2009-09-23 | MERCK PATENT GmbH | Anti-cancer antibodies with reduced complement fixation |
| SI2298815T1 (en) | 2005-07-25 | 2015-08-31 | Emergent Product Development Seattle, Llc | B-cell reduction using CD37-specific and CD20-specific binding molecules |
| ATE555125T1 (en) | 2005-12-30 | 2012-05-15 | Merck Patent Gmbh | INTERLEUKIN 12P40 VARIANTS WITH IMPROVED STABILITY |
| JP2009521912A (en) | 2005-12-30 | 2009-06-11 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング | Anti-CD19 antibody with reduced immunogenicity |
| NZ573646A (en) | 2006-06-12 | 2012-04-27 | Wyeth Llc | Single-chain multivalent binding proteins with effector function |
| WO2009126944A1 (en) | 2008-04-11 | 2009-10-15 | Trubion Pharmaceuticals, Inc. | Cd37 immunotherapeutic and combination with bifunctional chemotherapeutic thereof |
| AU2010238858A1 (en) | 2009-04-22 | 2011-12-08 | Merck Patent Gmbh | Antibody fusion proteins with modified FcRn binding sites |
| GB201403775D0 (en) | 2014-03-04 | 2014-04-16 | Kymab Ltd | Antibodies, uses & methods |
| JP7002446B2 (en) | 2015-09-21 | 2022-03-04 | アプティーボ リサーチ アンド デベロップメント エルエルシー | CD3 binding polypeptide |
| US9567399B1 (en) | 2016-06-20 | 2017-02-14 | Kymab Limited | Antibodies and immunocytokines |
| US11779604B2 (en) | 2016-11-03 | 2023-10-10 | Kymab Limited | Antibodies, combinations comprising antibodies, biomarkers, uses and methods |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ATE143052T1 (en) * | 1986-07-07 | 1996-10-15 | Centocor Inc | CHIMERIC MUINE-HUMAN IMMUNOGLOBULIN SPECIFIC FOR TUMOR-ASSOCIATED 17-1A ANTIGEN |
-
1991
- 1991-01-30 WO PCT/US1991/000633 patent/WO1991013166A1/en not_active Application Discontinuation
- 1991-01-30 EP EP91904780A patent/EP0517754A1/en not_active Ceased
- 1991-01-30 CA CA002077348A patent/CA2077348A1/en not_active Abandoned
- 1991-01-30 JP JP3505551A patent/JPH07507440A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| EP0517754A1 (en) | 1992-12-16 |
| WO1991013166A1 (en) | 1991-09-05 |
| JPH07507440A (en) | 1995-08-24 |
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