Chimeric antibody with specificity to human tumor antigen

A chimeric antibody with human constant region and murine variable region, having specificity to a human tumor antigen, methods of production, and uses. In particular, the present invention relates to a chimeric antibody specific for a human tumor antigen L6 and which mediates a potent antibody dependent cellular cytotoxicity against the tumor target cells.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to recombinant DNA methods of preparing an antibody 
with human tumor antigen specificity, genetic sequences coding therefor, 
as well as methods of obtaining such sequences. 
2. Background Art 
The application of cell-to-cell fusion for the production of monoclonal 
antibodies by Kohler and Milstein (Nature (London), 256:495, 1975) spawned 
a revolution in biology equal in impact to that from the invention of 
recombinant DNA cloning. Monoclonal antibodies produced from hybridomas 
are already widely used in clinical and basic scientific studies. 
Applications of human B cell hybridoma-produced monoclonal antibodies hold 
great promise for the treatment of cancer, viral and microbial infections, 
B cell immunodeficiencies with diminished antibody production, and other 
diseases and disorders of the immune system. 
Unfortunately, a number of obstacles exist with respect to the development 
of human monoclonal antibodies. This is especially true when attempting to 
develop monoclonal antibodies which define human tumor antigens for the 
diagnosis and treatment of cancer. Many of these tumor antigens are not 
recognized as foreign antigens by the human immune system, therefore, 
these antigens may not be immunogenic in man. By contrast, those human 
tumor antigens which are immunogenic in mice can be used for the 
production of mouse monoclonal antibodies that specifically recognize the 
human antigen and which may be used therapeutically in humans. However, 
repeated injections of "foreign" antibodies, such as a mouse antibody, in 
humans, can lead to harmful hypersensitivity reactions as well as 
increased rate of clearance of the circulating antibody molecules so that 
the antibodies do not reach their target site. 
Another problem faced by immunologists is that most human monoclonal 
antibodies obtained in cell culture are of the IgM type. When it is 
desirable to obtain human monoclonals of the IgG type, however, it has 
been necessary to use such techniques as cell sorting, to identify and 
isolate the few cells which are producing antibodies of the IgG or other 
type from the majority producing antibodies of the IgM type. A need 
therefore exists for an efficient method of switching antibody classes, 
for any given antibody of a predetermined or desired antigenic 
specificity. 
The present invention bridges both the hybridoma and genetic engineering 
technologies to provide a quick and efficient method, as well as products 
derived therefrom, for the production of a chimeric human/non-human 
antibody. 
The chimeric antibodies of the present invention embody a combination of 
the advantageous characteristics of monoclonal antibodies derived from 
mouse-mouse hybridomas and of human monoclonal antibodies. The chimeric 
monoclonal antibodies, like mouse monoclonal antibodies, can recognize and 
bind to a human target antigen; however, unlike mouse monoclonal 
antibodies, the species-specific properties of the chimeric antibodies 
will avoid the inducement of harmful hypersensitivity reactions and will 
allow for resistance to clearance when used in humans in vivo. Moreover, 
using the methods disclosed in the present invention, any desired antibody 
isotype can be conferred upon a particular antigen combining site. 
INFORMATION DISCLOSURE STATEMENT* 
Approaches to the problem of producing chimeric antibodies have been 
published by various authors. 
FNT *Note: The present Information Disclosure Statement is subject to the 
provisions of 37 C.F.R. 1.97(b). In addition, Applicants reserve the right 
to demonstrate that their invention was made prior to any one or more of 
the mentioned publications. 
Morrison, S. L. et al., Proc. Natl. Acad. Sci., U.S.A., 81:6851-6855 
(November 1984), describe the production of a mouse-human antibody 
molecule of defined antigen binding specificity, produced by joining the 
variable region genes of a mouse antibody-producing myeloma cell line with 
known antigen binding specificity to human immunoglobulin constant region 
genes using recombinant DNA techniques. Chimeric genes were constructed, 
wherein the heavy chain variable region exon from the myeloma cell line 
S107 were joined to human IgG1 or IgG2 heavy chain constant region exons, 
and the light chain variable region exon from the same myeloma to the 
human kappa light chain exon. These genes were transfected into mouse 
myeloma cell lines and. Transformed cells producing chimeric mouse-human 
antiphosphocholine antibodies were thus developed. 
Morrison, S. L. et al. , European Patent Publication No. 173494 (published 
Mar. 5, 1986), disclose chimeric "receptors" (e.g. antibodies) having 
variable regions derived from one species and constant regions derived 
from another. Mention is made of utilizing cDNA cloning to construct the 
genes, although no details of cDNA cloning or priming are shown. (see pp 
5, 7 and 8). 
Boulianne, G. L. et al., Nature, 312: 643 (Dec. 13, 1984), also produced 
antibodies consisting of mouse variable regions joined to human constant 
regions. They constructed immunoglobulin genes in which the DNA segments 
encoding mouse variable regions specific for the hapten trinitrophenyl 
(TNP) were joined to segments encoding human mu and kappa constant 
regions. These chimeric genes were expressed as functional TNP binding 
chimeric IgM. 
For a commentary on the work of Boulianne et al. and Morrison et al., see 
Munro, Nature, 312:597 (Dec. 13, 1984), Dickson, Genetic Engineering News, 
5, No. 3 (March 1985), or Marx, Science, 229:455 (August 1985). 
Neuberger, M. S. et al., Nature, 314:268 (Mar. 25, 1985), also constructed 
a chimeric heavy chain immunoglobulin gene in which a DNA segment encoding 
a mouse variable region specific for the hapten 
4-hydroxy-3-nitrophenacetyl (NP) was joined to a segment encoding the 
human epsilon region. When this chimeric gene was transfected into the 
J558L cell line, an antibody was produced which bound to the NP hapten and 
had human IgE properties. 
Neuberger, M. S. et al., have also published work showing the preparation 
of cell lines that secrete hapten-specific antibodies in which the Fc 
portion has been replaced either with an active enzyme moiety (Williams, 
G. and Neuberger, M. S. Gene 43:319, 1986) or with a polypeptide 
displaying c-myc antigenic determinants (Nature, 312:604, 1984). 
Neuberger, M. et al., PCT Publication WO 86/01533, (published Mar. 13, 
1986) also disclose production of chimeric antibodies (see p. 5) and 
suggests, among the technique's many uses the concept of "class switching" 
(see p. 6). 
Taniguchi, M., in European Patent Publication No. 171 496 (published Feb. 
19, 1985) discloses the production of chimeric antibodies having variable 
regions with tumor specificity derived from experimental animals, and 
constant regions derived from human. The corresponding heavy and light 
chain genes are produced in the genomic form, and expressed in mammalian 
cells. 
Takeda, S. et al., Nature, 314:452 (Apr. 4, 1985) have described a 
potential method for the construction of chimeric immunoglobulin genes 
which have intron sequences removed by the use of a retrovirus vector. 
However, an unexpected splice donor site caused the deletion of the V 
region leader sequence. Thus, this approach did not yield complete 
chimeric antibody molecules. 
Cabilly, S. et al., Proc. Natl. Acad. Sci. U.S.A., 81:3273-3277 (June 
1984), describe plasmids that direct the synthesis in E. coli of heavy 
chains and/or light chains of anti-carcinoembryonic antigen (CEA) 
antibody. Another plasmid was constructed for expression of a truncated 
form of heavy chain (Fd) fragment in E. coli. Functional CEA-binding 
activity was obtained by in vitro reconstitution, in E. coli extracts, of 
a portion of the heavy chain with light chain. 
Cabilly, S., et al., European Patent Publication 125023 (published Nov. 14, 
1984) describes chimeric immunoglobulin genes and their presumptive 
products as well as other modified forms. On pages 21, 28 and 33 it 
discusses cDNA cloning and priming. 
Boss, M. A., European Patent Application 120694 (published Oct. 3, 1984) 
shows expression in E. coli of non-chimeric immunoglobulin chains with 
4-nitrophenyl specificity. There is a broad description of chimeric 
antibodies but no details (see p. 9). 
Wood, C. R. et al., Nature, 314: 446 (April, 1985) describe plasmids that 
direct the synthesis of mouse anti-NP antibody proteins in yeast. Heavy 
chain mu antibody proteins appeared to be glycosylated in the yeast cells. 
When both heavy and light chains were synthesized in the same cell, some 
of the protein was assembled into functional antibody molecules, as 
detected by anti-NP binding activity in soluble protein prepared from 
yeast cells. 
Alexander, A. et al., Proc. Nat. Acad. Sci. U.S.A., 79:3260-3264 (1982), 
describe the preparation of a cDNA sequence coding for an abnormally short 
human Ig gamma heavy chain (OMM gamma.sup.3 HCD serum protein) containing 
a 19- amino acid leader followed by the first 15 residues of the V region. 
An extensive internal deletion removes the remainder of the V and the 
entire C.sub.H 1 domain. This is cDNA coding for an internally deleted 
molecule. 
Dolby, T. W. et al., Proc. Natl. Acad. Sci. U.S.A., 77:6027-6031 (1980), 
describe the preparation of a cDNA sequence and recombinant plasmids 
containing the same coding for mu and kappa human immunoglobulin 
polypeptides. One of the recombinant DNA molecules contained codons for 
part of the CH.sub.3 constant region domain and the entire 3noncoding 
sequence. 
Seno, M. et al., Nucleic Acids Research, 11:719-726 (1983), describe the 
preparation of a cDNA sequence and recombinant plasmids containing the 
same coding for part of the variable region and all of the constant region 
of the human IgE heavy chain (epsilon chain). 
Kurokawa, T. et al., ibid, 11: 3077-3085 (1983), show the construction, 
using cDNA, of three expression plasmids coding for the constant portion 
of the human IgE heavy chain. 
Liu, F. T. et al., Proc. Nat. Acad. Sci., U.S.A., 81:5369-5373 (September 
1984), describe the preparation of a cDNA sequence and recombinant 
plasmids containing the same encoding about two-thirds of the CH.sub.2, 
and all of the C.sub.H 3 and C.sub.H 4 domains of human IgE heavy chain. 
Tsujimoto, Y. et al., Nucleic Acids Res., 2:8407-8414 (November 1984), 
describe the preparation of a human V lambda cDNA sequence from an Ig 
lambda-producing human Burkitt lymphoma cell line, by taking advantage of 
a cloned constant region gene as a primer for cDNA synthesis. 
Murphy, J., PCT Publication WO 83/03971 (published Nov. 24, 1983) discloses 
hybrid proteins made of fragments comprising a toxin and a cell-specific 
ligand (which is suggested as possibly being an antibody). 
Tan, et al., J. Immunol. 135:8564 (November, 1985), obtained expression of 
a chimeric human-mouse immunoglobulin genomic gene after transfection into 
mouse myeloma cells. 
Jones, P. T., et al., Nature 321:552 (May 1986) constructed and expressed a 
genomic construct where CDR domains of variable regions from a mouse 
monoclonal antibody were used to substitute for the corresponding domains 
in a human antibody. 
Sun, L. K., et al., Hybridoma 5 suppl. 1 S17 (1986), describes a chimeric 
human/mouse antibody with potential tumor specificity. The chimeric heavy 
and light chain genes are genomic constructs and expressed in mammalian 
cells. 
Sahagan et al., J. Immun. 137:1066-1074 (August 1986) describe a chimeric 
antibody with specificity to a human tumor associated antigen, the genes 
for which are assembled from genomic sequences. 
For a recent review of the field see also Morrison, S. L., Science 229: 
1202-1207 (Sep. 20, 1985) and Oi, V. T., et al., BioTechniques 4:214 
(1986). 
The Oi, et al., paper is relevant as it argues that the production of 
chimeric antibodies from cDNA constructs in yeast and/or bacteria is not 
necessarily advantageous. 
See also Commentary on page 835 in Biotechnology 4 (1986). 
SUMMARY OF THE INVENTION 
The invention provides a genetically engineered chimeric antibody of 
desired variable region specificity and constant region properties, 
through gene cloning and expression of light and heavy chains. The cloned 
immunoglobulin gene products can be produced by expression in genetically 
engineered cells. 
The application of oligodeoxynucleotide synthesis, recombinant DNA cloning, 
and production of specific immunoglobulin chains in various procaryotic 
and eucaryotic cells provides a means for the large scale production of a 
chimeric human/mouse monoclonal antibody with specificity to a human tumor 
antigen. 
The invention provides cDNA sequences coding for immunoglobulin chains 
comprising a constant human region and a variable, non-human, region. The 
immunoglobulin chains can either be heavy or light. 
The invention provides gene sequences coding for immunoglobulin chains 
comprising a cDNA variable region of the desired specificity. These can be 
combined with genomic constant regions of human origin. 
The invention provides sequences as above, present in recombinant DNA 
molecules in vehicles such as plasmid vectors, capable of expression in 
desired prokaryotic or eukaryotic hosts. 
The invention provides hosts capable of producing, by culture, the chimeric 
antibodies and methods of using these hosts. 
The invention also provides individual chimeric immunoglobulin individual 
chains, as well as complete assembled molecules having human constant 
regions and variable regions with a human tumor antigen specificity, 
wherein both variable regions have the same binding specificity. 
Among other immunoglobulin chains and/or molecules provided by the 
invention are: 
(a) a complete functional, immunoglobulin molecule comprising: 
(i) two identical chimeric heavy chains comprising a variable region with a 
human tumor antigen specificity and human constant region and 
(ii) two identical all (i.e. non-chimeric) human light chains. 
(b) a complete, functional, immunoglobulin molecule comprising: 
(i) two identical chimeric heavy chains comprising a variable region as 
indicated, and a human constant region, and 
(ii) two identical all (i.e. non-chimeric) non-human light chains. 
(c) a monovalent antibody, i.e., a complete, functional immunoglobulin 
molecule comprising: 
(i) two identical chimeric heavy chains comprising a variable region as 
indicated, and a human constant region, and 
(ii) two different light chains, only one of which has the same specificity 
as the variable region of the heavy chains. The resulting antibody 
molecule binds only to one end thereof and is therefore incapable of 
divalent binding. 
Genetic sequences, especially cDNA sequences, coding for the aforementioned 
combinations of chimeric chains or of non-chimeric chains are also 
provided herein. 
The invention also provides for a genetic sequence, especially a cDNA 
sequence, coding for the variable region of desired specificity of an 
antibody molecule heavy and/or light chain, operably linked to a sequence 
coding for a polypeptide different than an immunoglobulin chain (e.g., an 
enzyme). These sequences can be assembled by the methods of the invention, 
and expressed to yield mixed-function molecules. 
The use of cDNA sequences is particularly advantageous over genomic 
sequences (which contain introns), in that cDNA sequences can be expressed 
in bacteria or other hosts which lack appropriate RNA splicing systems.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
INTRODUCTION 
Generally, antibodies are composed of two light and two heavy chain 
molecules. Light and heavy chains are divided into domains of structural 
and functional homology. The variable domains of both the light (V.sub.L) 
and the heavy (V.sub.H) chains determine recognition and specificity. The 
constant region domains of light (C.sub.L) and heavy (C.sub.H) chains 
confer important biological properties such as antibody chain association, 
secretion, transplacental mobility, complement binding, and the like. 
A complex series of events leads to immunoglobulin gene expression in B 
cells. The V region gene sequences conferring antigen specificity and 
binding are located in separate germ line gene segments called V.sub.H, D 
and J.sub.H ; or V.sub.L and J.sub.L. These gene segments are joined by 
DNA rearrangements to form the complete V regions expressed in heavy and 
light chains respectively (FIG. 1). The rearranged, joined (V.sub.L 
--J.sub.L and V.sub.H --D--J.sub.H) V segments then encode the complete 
variable regions or antigen binding domains of light and heavy chains, 
respectively. 
DEFINITIONS 
Certain terms and phrases are used throughout the specification and claims. 
The following definitions are provided for purposes of clarity and 
consistency. 
1. Expression vector--a plasmid DNA containing necessary regulatory signals 
for the synthesis of mRNA derived from any gene sequence, inserted into 
the vector. 
2. Module vector--a plasmid DNA containing a constant or variable region 
gene module. 
3. Expression plasmid--an expression vector that contains an inserted gene, 
such as a chimeric immunoglobulin gene. 
4. Gene cloning--synthesis of a gene, insertion into DNA vectors, 
identification by hybridization, sequence analysis and the like. 
5. Transfection--the transfer of DNA into mammalian cells. 
GENETIC PROCESSES AND PRODUCTS 
The invention provides a novel approach for the cloning and production of a 
human/mouse chimeric antibody with specificity to a human tumor antigen. 
The antigen is that bound by the monoclonal antibody described in Cancer 
Res. 46:3917-3923 (1986), and in Proc. Nat. Acad. Sci. U.S.A. 83:7059-7063 
(1986). The hybridoma secreting this monoclonal antibody was deposited at 
the American Type Culture Collection, Rockville, Maryland on Dec. 6, 1984 
with accession No. NB 8677. 
The method of production combines five elements: 
(1) Isolation of messenger RNA (mRNA) from the mouse B cell hybridoma line 
producing the monoclonal antibody, cloning and cDNA production therefrom; 
(2) Preparation of Universal Immunoglobulin Gene (UIG) oligonucleotides, 
useful as primers and/or probes for cloning of the variable region gene 
segments in the light and heavy chain mRNA from the hybridoma cell line, 
and cDNA production therefrom; 
(3) Preparation of constant region gene segment modules by cDNA preparation 
and cloning, or genomic gene preparation and cloning; 
(4) Construction of complete heavy or light chain coding sequences by 
linkage of the cloned specific immunoglobulin variable region gene 
segments of part (2) above to cloned human constant region gene segment 
modules; 
(5) Expression and production of light and heavy chains in selected hosts, 
including prokaryotic and eukaryotic cells, either in separate 
fermentations followed by assembly of antibody molecules in vitro., or 
through production of both chains in the same cell. 
One common feature of all immunoglobulin light and heavy chain genes and 
the encoded messenger RNAs is the so-called J region (i.e. joining region, 
see FIG. 1). Heavy and light chain J regions have different sequences, but 
a high degree of sequence homology exists (greater than 80%) especially 
near the constant region, within the heavy J.sub.H regions or the kappa 
light chain J regions. This homology is exploited in this invention and 
consensus sequences of light and heavy chain J regions were used to design 
oligonucleotides (designated herein as UIGs) for use as primers or probes 
for cloning immunoglobulin light or heavy chain mRNAs or genes (FIG. 3). 
Depending on the sequence of a particular UIG, it may be capable of 
hybridizing to all immunoglobulin mRNAs or genes containing a single 
specific J sequence. Another utility of a particular UIG probe may be 
hybridization to light chain or heavy chain mRNAs of a specific constant 
region, such as UIG-MJK which detects all mouse J.sub.K containing 
sequences (FIGS. 2A and 2B). UIG design can also include a sequence to 
introduce a restriction enzyme site into the cDNA copy of an 
immunoglobulin gene (see FIG. 3). The invention may, for example, utilize 
chemical gene synthesis to generate the UIG probes for the cloning and 
modification of V regions in immunoglobulin mRNA. On the other hand, 
oligonucleotides can be synthesized to recognize individually, the less 
conserved 5'-region of the J regions as a diagnostic aid in identifying 
the particular J region present in the immunoglobulin mRNA. 
On the other hand oligonucleotides can be synthesized to recognize 
individually the less conserved 5' region of the J region as a diagnostic 
aid in identifying the particular J region present in the Ig mRNA. 
A multi-step procedure is utilized for generating complete V+C region cDNA 
clones from the hybridoma cell light and heavy chain mRNAs. First, the 
complementary strand of oligo (dT)-primed cDNA is synthesized, and this 
double-stranded cDNA is cloned in appropriate cDNA cloning vectors such as 
pBR322 (Gubler and Hoffman, Gene, 25: 263 (1983)). Clones are screened by 
hybridization with UIG oligonucleotide probes. Positive heavy and light 
chain clones identified by this screening procedure are mapped and 
sequenced to select those containing V region and leader coding sequences. 
In vitro mutagenesis including, for example, the use of UIG 
oligonucleotides, is then used to engineer desired restriction enzyme 
cleavage sites. 
An alternative method is to use synthetic UIG oligonucleotides as primers 
for the synthesis of cDNA. 
Second, cDNA constant region module vectors are prepared from human cells. 
These cDNA clones are modified, when necessary, by site-directed 
mutagenesis to place a restriction site at the analogous position in the 
human sequence or at another desired location near a boundary of the 
constant region. An alternative method utilizes genomic C region clones as 
the source for C region module vectors. 
Third, cloned V region segments generated as above are excised and ligated 
to light or heavy chain C region module vectors. For example, one can 
clone the complete human kappa light chain C region and the complete human 
gamma.sub.1 C region. In addition, one can modify the human gamma.sub.1 
region to introduce a termination codon and thereby obtain a gene sequence 
which encodes the heavy chain portion of an Fab molecule. 
The coding sequences having operationally linked V and C regions are then 
transferred into appropriate expression vehicles for expression in 
appropriate hosts, prokaryotic or eukaryotic. Operationally linked means 
in-frame joining of coding sequences to derive a continuously translatable 
gene sequence without alterations or interruptions of the triplet reading 
frame. 
One particular advantage of using cDNA genetic sequences in the present 
invention is the fact that they code continuously for immunoglobulin 
chains, either heavy or light. By "continuously" is meant that the 
sequences do not contain introns (i.e. are not genomic sequences, but 
rather, since derived from mRNA by reverse transcription, are sequences of 
contiguous exons). This characteristic of the cDNA sequences provided by 
the invention allows them to be expressible in prokaryotic hosts, such as 
bacteria, or in lower eukaryotic hosts, such as yeast. 
Another advantage of using cDNA cloning method is the ease and simplicity 
of obtaining variable region gene modules. 
The terms "constant" and "variable" are used functionally to denote those 
regions of the immunoglobulin chain, either heavy or light chain, which 
code for properties and features possessed by the variable and constant 
regions in natural non-chimeric antibodies. As noted, it is not necessary 
for the complete coding region for variable or constant regions to be 
present, as long as a functionally operating region is present and 
available. 
Expression vehicles include plasmids or other vectors. Preferred among 
these are vehicles carrying a functionally complete human constant heavy 
or light chain sequence having appropriate restriction sites engineered so 
that any variable heavy or light chain sequence with appropriate cohesive 
ends can be easily inserted thereinto. Human constant heavy or light chain 
sequence-containing vehicles are thus an important embodiment of the 
invention. These vehicles can be used as intermediates for the expression 
of any desired complete heavy or light chain in any appropriate host. 
One preferred host is yeast. Yeast provides substantial advantages for the 
production of immunoglobulin light and heavy chains. Yeasts carry out 
post-translational peptide modifications including glycosylation. A number 
of recombinant DNA strategies now exist which utilize strong promoter 
sequences and high copy number plasmids which can be used for overt 
production of the desired proteins in yeast. Yeast recognizes leader 
sequences on cloned mammalian gene products and secretes peptides bearing 
leader sequences (i.e. prepeptides) (Hitzman, et al., 11th International 
Conference on Yeast, Genetics and Molecular Biology, Montpelier, France, 
Sep. 13-17, 1982). 
Yeast gene expression systems can be routinely evaluated for the level of 
heavy and light chain production, protein stability, and secretion. Any of 
a series of yeast gene expression systems incorporating promoter and 
termination elements from the actively expressed genes coding for 
glycolytic enzymes produced in large quantities when yeasts are grown in 
mediums rich in glucose can be utilized. Known glycolytic genes can also 
provide very efficient transcription control signals. For example, the 
promoter and terminator signals of the iso-1-cytochrome C (CYC-1) gene can 
be utilized. 
The following approach can be taken for evaluating optimal expression 
plasmids for the expression of cloned immunoglobulin cDNAs in yeast. 
(1) The cloned immunoglobulin DNA linking V and C regions is attached to 
different transcription promoters and terminator DNA fragments; 
(2) The chimeric genes are placed on yeast plasmids (see, for example, 
Broach, J. R. in Methods in Enzymology--Vol. 101:307 ed. Wu, R. et al., 
1983)); 
(3) Additional genetic units such as a yeast leader peptide may be included 
on immunoglobulin DNA constructs to obtain antibody secretion. 
(4) A portion of the sequence, frequently the first 6 to 20 codons of the 
gene sequence may be modified to represent preferred yeast codon usage. 
(5) The chimeric genes are placed on plasmids used for integration into 
yeast chromosomes. 
The following approaches can be taken to simultaneously express both light 
and heavy chain genes in yeast. 
(1) The light and heavy chain genes are each attached to a yeast promoter 
and a terminator sequence and placed on the same plasmid. This plasmid can 
be designed for either autonomous replication in yeast or integration at 
specific sites in the yeast chromosome. 
(2) The light and heavy chain genes are each attached to a yeast promoter 
and terminator sequence on separate plasmids containing different 
selective markers. For example, the light chain gene can be placed on a 
plasmid containing the trp1 gene as a selective marker, while the heavy 
chain gene can be placed on a plasmid containing ura3 as a selective 
marker. The plasmids can be designed for either autonomous replication in 
yeast or integration at specific sites in yeast chromosomes. A yeast 
strain defective for both selective markers is either simultaneously or 
sequentially transformed with the plasmid containing the light chain gene 
and with the plasmid containing the heavy chain gene. 
(3) The light and heavy chain genes are each attached to a yeast promoter 
and terminator sequence on separate plasmids each containing different 
selective markers as described in (2) above. A yeast mating type "a" 
strain defective in the selective markers found on the light and heavy 
chain expression plasmids (trp1 and ura3 in the above example) is 
transformed with the plasmid containing the light chain gene by selection 
for one of the two selective markers (trp1 in the above example). A yeast 
mating type "alpha" strain defective in the same selective markers as the 
"a" strain (i.e. trp1 and ura3 as examples) is transformed with a plasmid 
containing the heavy chain gene by selection for the alternate selective 
marker (i.e. ura3 in the above example). The "a" strain containing the 
light chain plasmid (phenotype: Trp.sup.+ Ura.sup.- in the above example) 
and the strain containing the heavy chain plasmid (pheno-type: Trp.sup.- 
Ura.sup.+ in the above example) are mated and diploids are selected which 
are prototrophic for both of the above selective markers (Trp.sup.+ 
Ura.sup.+ in the above example). 
Among bacterial hosts which may be utilized as transformation hosts, E. 
coli K12 strain 294 (ATCC 31446) is particularly useful. Other microbial 
strains which may be used include E. coli X1776 (ATCC 31537). The 
aforementioned strains, as well as coli W3110 (ATCC 27325) and other 
enterobacteria such as Salmonella typhimurium or Serratia marcescens, and 
various Pseudomonas species may be used. 
In general, plasmid vectors containing replicon and control sequences which 
are derived from species compatible with a host cell are used in 
connection with these hosts. The vector ordinarily carries a replication 
site, as well as specific genes which are capable of providing phenotypic 
selection in transformed cells. For example, E. coli is readily 
transformed using pBR322, a plasmid derived from an E. coli species 
(Bolivar, et al., Gene, 2:95 (1977)). pBR322 contains genes for ampicillin 
and tetracycline resistance, and thus provides easy means for identifying 
transformed cells. The pBR322 plasmid or other microbial plasmids must 
also contain, or be modified to contain, promoters which can be used by 
the microbial organism for expression of its own proteins. Those promoters 
most commonly used in recombinant DNA construction include the 
beta-lactamase (penicillinase) and lactose (beta-galactosidase) promoter 
systems (Chang et al., Nature, 275:615 (1978); Itakura et al., Science, 
198:1056 (1977)); and tryptophan promoter systems (Goeddel et al., Nucleic 
Acids Research, 8:4057 (1980); EPO Publication No. 0036776). While these 
are the most commonly used, other microbial promoters have been discovered 
and utilized. 
For example, a genetic construct for any heavy or light chimeric 
immunoglobulin chain can be placed under the control of the leftward 
promoter of bacteriophage lambda (P.sub.L). This promoter is one of the 
strongest known promoters which can be controlled. Control is exerted by 
the lambda repressor, and adjacent restriction sites are known. 
The expression of the immunoglobulin chain sequence can also be placed 
under control of other regulatory sequences which may be "homologous" to 
the organism in its untransformed state. For example, lactose dependent E. 
coli chromosomal DNA comprises a lactose or lac operon which mediates 
lactose digestion by elaborating the enzyme beta-galactosidase. The lac 
control elements may be obtained from bacteriophage lambda pLAC5, which is 
infective for E. coli. The lac promoter-operator system can be induced by 
IPTG. 
Other promoter/operator systems or portions thereof can be employed as 
well. For example, arabinose, colicine El, galactose, alkaline 
phosphatase, tryptophan, xylose, tac, and the like can be used. 
Other preferred hosts are mammalian cells, grown in vitro in tissue 
culture, or in vivo in animals. Mammalian cells provide post-translational 
modifications to immunoglobulin protein molecules including leader peptide 
removal, correct folding and assembly of heavy and light chains, proper 
glycosylation at correct sites, and secretion of functional antibody 
protein. 
Mammalian cells which may be useful as hosts for the production of antibody 
proteins include cells of lymphoid origin, such as the hybridoma 
Sp2/0-Ag14 (ATCC CRL 1581) or the myeloma P3X63Ag8 (ATCC TIB 9), and its 
derivatives. Others include cells of fibroblast origin, such as Vero (ATCC 
CRL 81) or CHO- K1 (ATCC CRL 61). 
Several possible vector systems are available for the expression of cloned 
heavy chain and light chain genes in mammalian cells. One class of vectors 
relies upon the integration of the desired gene sequences into the host 
cell genome. Cells which have stably integrated DNA can be selected by 
simultaneously introducing drug resistance genes such as E. coli gpt 
(Mulligan, R. C. and Berg, P., Proc. Natl. Acad. Sci., U.S.A., 78: 2072 
(1981)) or Tn5 neo (Southern, P. J. and Berg, P., J. Mol. Appl. Genet., 
1:327 (1982)). The selectable marker gene can be either linked to the DNA 
gene sequences to be expressed, or introduced into the same cell by 
co-transfection (Wigler, M. et al., Cell, 16: 77 (1979)). A second class 
of vectors utilizes DNA elements which confer autonomously replicating 
capabilities to an extrachromosomal plasmid. These vectors can be derived 
from animal viruses, such as bovine papillomavirus (Sarver, N. et al., 
Proc. Natl. Acad. Sci. U.S.A., 79:7147 (1982)), polyoma virus (Deans, R. 
J. et al., Proc. Natl. Acad. Sci. U.S.A., 81:1292 (1984)), or SV40 virus 
(Lusky, M. and Botchan, M., Nature, 293: 79 (1981)). 
Since an immunoglobulin cDNA is comprised only of sequences representing 
the mature mRNA encoding an antibody protein, additional gene expression 
elements regulating transcription of the gene and processing of the RNA 
are required for the synthesis of immunoglobulin mRNA. These elements may 
include splice signals, transcription promoters, including inducible 
promoters enhancers, and termination signals. cDNA expression vectors 
incorporating such elements include those described by Okayama, H. and 
Berg, P., Mol. Cell Biol., 3:280 (1983); Cepko, C. L. et al., Cell, 
37:1053 (1984); and Kaufman, R. J. , Proc. Natl. Acad. Sci., U.S.A., 
82:689 (1985). 
An additional advantage of mammalian cells as hosts is their ability to 
express chimeric immunoglobulin genes which are derived from genomic 
sequences. Thus, mammalian cells may express chimeric immunoglobulin genes 
which are comprised of a variable region cDNA module plus a constant 
region which is composed in whole or in part of genomic sequences. Several 
human constant region genomic clones have been described (Ellison, J. W. 
et al., Nucl. Acids Res., 10:4071 (1982), or Max, E. et al., Cell, 29:691 
(1982)). The use of such genomic sequences may be convenient for the 
simultaneous introduction of immunoglobulin enhancers, splice signals, and 
transcription termination signals along with the constant region gene 
segment. 
Different approaches can be followed to obtain complete H.sub.2 L.sub.2 
antibodies. 
First, one can separately express the light and heavy chains followed by in 
vitro assembly of purified light and heavy chains into complete H.sub.2 
L.sub.2 IgG antibodies. The assembly pathways used for generation of 
complete H.sub.2 L.sub.2 IgG molecules in cells have been extensively 
studied (see, for example, Scharff, M., Harvey Lectures, 69:125 (1974)). 
In vitro reaction parameters for the formation of IgG antibodies from 
reduced isolated light and heavy chains have been defined by Beychok, S., 
Cells of Immunoglobulin Synthesis, Academic Press, New York, page 69, 
1979. 
Second, it is possible to co-express light and heavy chains in the same 
cells to achieve intracellular association and linkage of heavy and light 
chains into complete H.sub.2 L.sub.2 IgG antibodies. The co-expression can 
occur by using either the same or different plasmids in the same host. 
POLYPEPTIDE PRODUCTS 
The invention provides "chimeric" immunoglobulin chains, either heavy or 
light. A chimeric chain contains a constant region substantially similar 
to that present in of a natural human immunoglobulin, and a variable 
region having the desired antigenic specificity of the invention, i.e., to 
the specified human tumor antigen. 
The invention also provides immunoglobulin molecules having heavy and light 
chains associated so that the overall molecule exhibits any desired 
binding and recognition properties. Various types of immunoglobulin 
molecules are provided: monovalent, divalent, molecules with chimeric 
heavy chains and non-chimeric light chains, or molecules with the 
invention's variable binding domains attached to moieties carrying desired 
functions. 
Antibodies having chimeric heavy chains of the same or different variable 
region binding specificity and non-chimeric (i.e., all human or all 
non-human) light, chains, can be prepared by appropriate association of 
the needed polypeptide chains. These chains are individually prepared by 
the modular assembly methods of the invention. 
USES 
The antibodies of the invention having human constant region can be 
utilized for passive immunization, especially -in humans, without negative 
immune reactions such as serum sickness or anaphylactic shock. The 
antibodies can, of course, also be utilized in prior art immunodiagnostic 
assays and kits in detectably labelled form (e.g., enzymes, .sup.125 I, 
.sup.14 C, fluorescent labels, etc.), or in immobilized form (on polymeric 
tubes, beads, etc.), in labelled form for in vivo imaging, wherein the 
label can be a radioactive emitter, or an NMR contrasting agent such as a 
carbon-13 nucleus, or an X-ray contrasting agent, such as a heavy metal 
nucleus. The antibodies can also be used for in vitro localization of the 
antigen by appropriate labelling. 
The antibodies can be used for therapeutic purposes, by themselves, in 
complement mediated lysis, or coupled to toxins or therapeutic moieties, 
such as ricin, etc. 
Mixed antibody-enzyme molecules can be used for immunodiagnostic methods, 
such as ELISA. Mixed antibody-peptide effector conjugates can be used for 
targeted delivery of the effector moiety with a high degree of efficacy 
and specificity. 
Specifically, the chimeric antibodies of this invention can be used for any 
and all uses in which the murine L6 monoclonal antibody can be used, with 
the obvious advantage that the chimeric ones are compatible with the human 
body. 
Having now generally described the invention, the same will be further 
understood by reference to certain specific examples which are included 
herein for purposes of illustration only and are not intended to be 
limiting unless otherwise specified. 
EXPERIMENTAL 
Materials and Methods 
Tissue Culture Cell Lines 
The human cell lines GM2146 and GM1500 were obtained from the Human Mutant 
Cell Repository (Camden, New Jersey) and cultured in RPMI1640 plus 10% 
fetal bovine serum (M. A. Bioproducts). The cell line Sp2/0 was obtained 
from the American Type Culture Collection and grown in Dulbecco's Modified 
Eagle Medium (DMEM) plus 4.5 g/l glucose (M. A. Bioproducts) plus 10% 
fetal bovine serum (Hyclone, Sterile Systems, Logan, Utah). Media were 
supplemented with penicillin/streptomycin (Irvine Scientific, Irvine, 
California). 
Recombinant Plasmid and Bacteriophage DNAs 
The plasmids pBR322, pL1 and pUC12 were purchased from Pharmacia P-L 
Biochemicals (Milwaukee, Wisconsin). The plasmids pSV2-neo and pSV2-gpt 
were obtained from BRL (Gaithersburg, Maryland), and are available from 
the American Type Culture Collection (Rockville, Maryland). pHu-gamma-1 is 
a subclone of the 8.3 Kb HindIII to BamHI fragment of the human IgGl 
chromosomal gene. An isolation method for the human IgGl chromosomal gene 
is described by Ellison, J. W. et al., Nucl. Acids Res., 10:4071 (1982). 
M8alphaRX12 contains the 0.7 Kb XbaI to EcoRI fragment containing the 
mouse heavy chain enhancer from the J-C intron region of the M603 
chromosomal gene (Davis, M. et al., Nature, 283:733, 1979) inserted into 
M13mp10. DNA manipulations involving purification of plasmid DNA by 
buoyant density centrifugation, restriction endonuclease digestion, 
purification of DNA fragments by agarose gel electrophoresis, ligation and 
transformation of E. coli were as described by Maniatis, T. et al., 
Molecular Cloning: A Laboratory Manual, (1982) or other procedures. 
Restriction endonucleases and other DNA/RNA modifying enzymes were 
purchased from Boehringer-Mannheim (Indianapolis, Indiana), BRL, New 
England Biolabs (Beverly, Massachusetts) and Pharmacia P-L. 
Oligonucleotide Preparation 
Oligonucleotides were either synthesized by the triester method of Ito et 
al. (Nucl. Acids Res., 10:1755 (1982)), or were purchased from ELESEN, Los 
Angeles, California. Tritylated, deblocked oligonucleotides were purified 
on Sephadex-G50, followed by reverse-phase HPLC with a 0-25% gradient of 
acetonitrile in 10 mM triethylamine-acetic acid, pH 7.2, on a C18 
uBondapak column (Waters Associates). Detritylation was in 80% acetic acid 
for 30 min., followed by evaporation thrice. Oligonucleotides were labeled 
with [gamma-.sup.32 P]ATP plus T4 polynucleotide kinase. 
RNA Preparation and Analysis 
Total cellular RNA was prepared from tissue culture cells by the method of 
Auffray, C. and Rougeon, F. (Eur. J. Biochem., 107:303 (1980)) or 
Chirgwin, J. M. et al. (Biochemistry, 18:5294 (1979)). Preparation of 
poly(A).sup.+ RNA, methyl-mercury agarose gel electrophoresis, and 
"Northern" transfer to nitrocellulose were as described by Maniatis, T. et 
al., supra. Total cellular RNA or poly(A).sup.+ RNA was directly bound to 
nitrocellulose by first treating the RNA with formaldehyde (White, B. A. 
and Bancroft, F. C., J. Biol. Chem., 257:8569 (1982)). Hybridization to 
filterbound RNA was with nick-translated DNA fragments using conditions 
described by Margulies, D. H. et al. (Nature, 295:168 (1982)) or with 
.sup.32 P-labelled oligonucleotide using 4xSSC, 10X Denhardt's, 100 ug/ml 
salmon sperm DNA at 37.degree. C. overnight, followed by washing in 4xSSC 
at 37.degree. C. 
cDNA Preparation and Cloning 
Oligo-dT primed cDNA libraries were prepared from poly(A).sup.+ RNA from 
GM1500 and GM2146 cells by the methods of Land, H. et al. (Nucl. Acids 
Res., 9:2251 (1981)) and Gubler, V. and Hoffman, B. J., Gene, 25:263 
(1983), respectively. The cDNA libraries were screened by hybridization 
(Maniatis, T., supra) with .sup.32 P-labelled oligonucleotides using the 
procedure of de Lange et al. (Cell, 34:891 (1983)), or with 
nick-translated DNA fragments. 
Oligonucleotide Primer Extension and Cloning 
Poly(A).sup.+ RNA (20 ug) was mixed with 1.2 ug primer in 40 ul of 64 mM 
KCl. After denaturation at 90.degree. C. for 5 min. and then chilling in 
ice, 3 units Human Placental Ribonuclease Inhibitor (BRL) was added in 3 
ul of 1M Tris-HCl, pH 8.3. The oligonucleotide was annealed to the RNA at 
42.degree. C. for 15 minutes, then 12 ul of 0.05M DTT, 0.05M MgCl.sub.2, 
and 1 mM each of dATP, dTTP, dCTP, and dGTP was added. 2 ul of 
alpha-.sup.32 P-dATP (400 Ci/mmol, New England Nuclear) was added, 
followed by 3 ul of AMV reverse transcriptase (19 units/ul, Life 
Sciences). 
After incubation at 42.degree. C. for 105 min., 2 ul 0.5M EDTA and 50 ul 10 
mM Tris, 1 mM EDTA, pH 7.6 were added. Unincorporated nucleotides were 
removed by Sephadex G-50 spun column chromatography, and the RNA-DNA 
hybrid was extracted with phenol, then with chloroform, and precipitated 
with ethanol. Second strand synthesis, homopolymer tailing with dGTP or 
dCTP, and insertion into homopolymer tailed vectors was as described by 
Gubler and Hoffman, supra. 
Site-Directed Mutagenesis 
Single stranded M13 subclone DNA (1 ug) was combined with 20 ng 
oligonucleotide primer in 12.5 ul of Hin buffer (7 mM Tris-HCl, pH 7.6, 7 
mM MgCl.sub.2, 50 mM NaCl). After heating to 95.degree. C. in a sealed 
tube, the primer was annealed to the template by slowly cooling from 
70.degree. C. to 37.degree. C. for 90 minutes. 2 ul dNTPs (1 mM each), 1 
ul .sup.32 P-dATP (10 uCi), 1 ul DTT (0.1M) and 0.4 ul Klenow DNA PolI 
(2u, Boehringer Mannheim) were added and chains extended at 37.degree. C. 
for 30 minutes. To this was added 1 ul (10 ng) M13 reverse primer (New 
England Biolabs), and the heating/annealing and chain extension steps were 
repeated. The reaction was stopped with 2 ul of 0.5M EDTA, pH 8, plus 80 
ul of 10 mM Tris-HCl, pH 7.6, 1 mM EDTA. The products were phenol 
extracted and purified by Sephadex G-50 spun column chromatography and 
ethanol precipitated prior to restriction enzyme digestion and ligation to 
the appropriate vector. 
Transfection of Myeloma Tissue Culture Cells 
The electroporation method of Potter, H. et al. (Proc. Natl. Acad. Sci. 
U.S.A., 81:7161 (1984)) was used. After transfection, cells were allowed 
to recover in complete DMEM for 48-72 hours, then were seeded at 10,000 to 
50,000 cells per well in 96-well culture plates in the presence of 
selective medium. G418 (GIBCO) selection was at 0.8 mg/ml, mycophenolic 
acid (Calbiochem) was at 6 ug/ml plus 0.25 mg/ml xanthine, and HAT (Sigma) 
was at the standard concentration. 
Assays for Immunoglobulin Synthesis and Secretion 
Secreted immunoglobulin was measured directly from tissue culture cell 
supernatants. Cytoplasmic protein extract was prepared by vortexing 106 
cells in 160 ul of 1% NP40, 0.15M NaCl, 10 mM Tris, 1 mM EDTA, pH 7.6 and 
leaving the lysate at 0.degree. C., 15 minutes, followed by centrifugation 
at 10,000.times.g to remove insoluble debris. 
A double antibody sandwich ELISA (Voller, A. et al., in Manual of Clinical 
Immunology, 2nd Ed., Eds. Rose, N. and Friedman, H., pp. 359-371, 1980) 
using affinity purified antisera was used to detect specific 
immunoglobulins. For detection of human IgG, the plate-bound antiserum is 
goat anti-human IgG (KPL, Gaithersburg, Maryland) at 1/1000 dilution, 
while the peroxidase-bound antiserum is goat anti-human IgG (KPL or Tago, 
Burlingame) at 1/4000 dilution. For detection of human immunoglobulin 
kappa, the plate-bound antiserum is goat anti-human kappa (Tago) at 1/500 
dilution, while the peroxidase-bound antiserum is goat anti-human kappa 
(Cappel) at 1/1000 dilution. 
EXAMPLE 1 
A Chimeric Mouse-Human Immunoglobulin with Cancer Antigen Specificity 
(1) Antibody L6 
L6 monoclonal antibody (MAb) was obtained from a mouse which had been 
immunized with cells from a human lung carcinoma, after which spleen cells 
were hybridized with NS-1 mouse myeloma cells. The antibody binds to a 
previously not identified carbohydrate antigen which is expressed in large 
amounts at the surface of cells from most human carcinomas, including lung 
carcinomas (adeno, squamous), breast carcinomas, colon carcinomas and 
ovarian carcinomas, while the antigen is only present at trace levels in 
normal cells from the adult host. MAb L6 is an IgG2a and can mediate 
antibody dependent cellular cytotoxicity, ADCC, in the presence of human 
peripheral blood leukocytes as a source of effector cells, so as to lyse 
L6 positive tumor cells, and it can lyse L6 positive tumor cells in the 
presence of human serum as a source of complement; the lysis is detected 
as the release of .sup.51 Cr from labelled cells over a 4 hour incubation 
period. MAb L6 can localize to L6 positive tumors xenotransplanted onto 
nude mice, and it can inhibit the outgrowth of such tumors. MAb L 6 is 
described in Cancer Res. 46:3917-3923, 1986 (on MAb specificity) and in 
Proc. Natl. Acad. Sci. 83:7059-7063, 1986 (on MAb function). MAb L6 is 
also described in copending application Ser No. 776,321 filed Oct. 18, 
1985, and now U.S. Pat. No. 4,906,562, and Ser. No. 684,759 filed Dec. 21, 
1984, and now U.S. Pat. No. 4,935,495, the contents of each of which is 
fully incorporated by reference. 
(2) Identification of J Sequences in the Immunoglobulin mRNA of L6 
Frozen cells were thawed on ice for 10 minutes and then at room 
temperature. The suspension was diluted with 15 ml PBS and the cells were 
centrifuged down. They were resuspended, after washes in PBS, in 16 ml 3M 
LiCl, 6M urea and disrupted in a polytron shear. The preparation of mRNA 
and the selection of the poly(A+) fraction were carried out according to 
Auf-fray, C. and Rougeon, F., Eur. J. Biochem. 107:303, 1980. 
The poly (A+) RNA from L6 was hybridized individually with labeled J.sub.H 
1, J.sub.H 2, J.sub.H 3 and J.sub.H 4 oligonucleotides under conditions 
described by Nobrega et al. Anal. Biochem 131:141, 1983). The products 
were then subjected to electrophoresis in a 1.7% agarose-TBE gel. The gel 
was fixed in 10% TCA, blotted dry and exposed for autoradiography. The 
result showed that the L6 v.sub.H contains J.sub.H 2 sequences. 
For the analysis of the V.sub.K mRNA, the dot-blot method of White and 
Bancroft J. Biol. Chem. 257:8569, (1982) was used. Poly (A+) RNA was 
immobilized on nitrocellulose filters and was hybridized to labeled 
probe-oligonucleotides at 40.degree. in 4xSSC. These experiments show that 
L6 contains JK.sup.5 sequences. A faint hybridization to J.sub.K 2 was 
observed. 
(3) V Region cDNA Clones 
A library primed by oligo (dT) on L6 poly (A+) RNA was screened for kappa 
clones with a mouse C.sub.K region probe. From the L6 library, several 
clones were isolated. A second screen with a 5' J.sub.K 5 specific probe 
identified the L6 (J.sub.K 5) light-chain clones. Heavy chain clones of L6 
were isolated by screening with the J.sub.H 2 oligonucleotide. 
The heavy and light chain genes or gene fragments from the cDNA clones, pH 
3-6a and pL3-12a were inserted into M13 bacteriophage vectors for 
nucleotide sequence analysis. The complete nucleotide sequences of the 
variable region of these clones were determined (FIGS. 5A, 5B and 6) by 
the dideoxy chain termination method. These sequences predict V region 
amino acid compositions that agree well with the observed compositions, 
and predict peptide sequences which have been verified by direct amino 
acid sequencing of portions of the V regions. 
The nucleotide sequences of the cDNA clones show that they are 
immunoglobulin V region clones as they contain amino acid residues 
diagnostic of V domains (Kabat et al. , Sequences of Proteins of 
Immunological Interest; U.S. Dept of HHS, 1983). 
The L6 V.sub.H belongs to subgroup II. The cDNA predicts an N-terminal 
sequence of 24 amino acid residues identical to that of a known V.sub.H 
(45-165 CRI; Margolies et al. Mol. Immunol. 18:1065, 1981). The L6 V.sub.H 
has the J.sub.H 2 sequence. The L6 V.sub.L is from the V.sub.K -KpnI 
family (Nishi et al. Proc. Nat. Acd. Sci. U.S.A. 82:6399, 1985), and uses 
J.sub.K 5. The cloned L6 V.sub.L predicts an amino acid sequence which was 
confirmed by amino acid sequencing of peptides from the L6 light chain 
corresponding to residues 18-40 and 80-96. 
(4) In Vitro Mutagenesis to Engineer Restriction Enzyme Sites in the J 
Region for Joining to a Human C-Module and to Remove Oligo (dC) Sequences 
5' to the V Modules 
Both clones generated from priming with oligo (dT) L6 V.sub.K and L6 
V.sub.H need to be modified. For the L6 V.sub.K, the J-region mutagenesis 
primer JKHindIII, as shown in FIG. 6, was utilized. A human C.sub.K module 
derived from a cDNA clone was mutagenized to contain the HindIII sequence 
(see FIG. 4). The mutagenesis reaction was performed on M13 subclones of 
these genes. The frequency of mutant clones ranged from 0.5 to 1% of the 
plaques obtained. 
It had been previously observed that the oligo (dC) sequence upstream of 
the AUG codon in a V.sub.H chimeric gene interferes with proper splicing 
in one particular gene construct. It was estimated that perhaps as much as 
70% of the RNA transcripts had undergone the mis-splicing, wherein a 
cryptic 3' splice acceptor in the leader sequence was used. Therefore the 
oligo (dC) sequence upstream of the initiator AUG was removed in all of 
the clones. 
In one approach, an oligonucleotide was used which contains a SalI 
restriction site to mutagenize the L6 V.sub.K clone. The primer used for 
this oligonucleotide-directed mutagenesis is a 22-mer which introduces a 
SalI site between the oligo (dC) and the initiator met codon (FIGS. 9A and 
9B). 
In a different approach, the nuclease BAL-31 was used to chew away the 
oligo (dC) in the L6 V.sub.H clone pH 3-6a. The size of the deletion in 
two of the mutants obtained was determined by nucleotide sequencing and is 
shown in FIGS. 7A and 7B. In both of these mutuants (delta 4 and delta 
21), all of the oligo (dC) 5' to the coding region were deleted. 
These clones were then modified by oligonucleotide-directed mutagenesis 
with the MJH2-ApaI primer (FIGS. 7A and 7B). This 31-base primer 
introduces an ApaI site in the mouse C.sub.H gene at a position analogous 
to an existing ApaI site in human Cgamma1 cDNA gene module. The primer 
introduces the appropriate codons for the human C gamma 1 gene. The 
chimeric heavy chain gene made by joining the mutagenized mouse V.sub.H 
gene module to a human C.sub.H module thus encodes a chimeric protein 
which contains no human amino acids for the entire V.sub.H region. 
The human C gamma 1 gene module is a cDNA derived from GM2146 cells (Human 
Genetic Mutant Cell Repository, Newark, New Jersey). This C gamma 1 gene 
module was previously combined with a mouse V.sub.H gene module to form 
the chimeric expression plasmid pING2012E. 
(5) L6 Chimeric Expression Plasmids 
L6 chimeric heavy chain expression plasmids were derived from the 
replacement of the V.sub.H module pING2012E with the V.sub.H modules of 
mutants delta 21 and delta 4 to give the expression plasmids pING2111 and 
pING2112 (FIGS. 7A and 7B). These plasmids direct the synthesis of 
chimeric L6 heavy chain when transfected into mammalian cells. 
For the L6 light chain chimeric gene, the SalI to HindIII fragment of the 
mouse V.sub.K module was joined to the human C.sub.K module by the 
procedure outlined in FIG. 8, forming pING2119. Replacement of the neo 
sequence with the E. coli gpt gene derived from pSV2-gpt resulted in 
pING2120, which expressed L6 chimeric light chain and confers mycophenolic 
acid resistance when transfected into mammalian cells. 
The inclusion of both heavy and light chain chimeric genes in the same 
plasmid allows for the introduction into transfected cells of a 1:1 gene 
ratio of heavy and light chain genes leading to a balanced gene dosage. 
This may improve expression and decrease manipulations of transfected 
cells for optimal chimeric antibody expression. For this purpose, the DNA 
fragments derived from the chimeric heavy and light chain genes of 
pING2111 and pING2119 were combined into the expression plasmid pING2114 
(FIGS. 9A and 9B). This expression plasmid contains a selectable neo.sup.R 
marker and separate transcription units for each chimeric gene, each 
including a mouse heavy chain enhancer. 
The modifications and V-C joint regions of the L6 chimeric genes are 
summarized in FIG. 10. 
(6) Stable Transfection of Mouse Lymphoid Cells for the Production of 
Chimeric Antibody 
Electroporation was used (Potter et al. supra; Toneguzzo et al. Mol. Cell 
Biol. 6:703 1986) for the introduction of L6 chimeric expression plasmid 
DNA into mouse Sp2/0 cells. The electroporation technique gave a 
transfection frequency of 1-10.times.10.sup.-5 for the Sp2/0 cells. 
The two gene expression plasmid pING2114 was linearized by digestion with 
AatII restriction endonuclease and transfected into Sp2/0 cells, giving 
approximately fifty G418 resistant clones which were screened for human 
heavy and light chain synthesis. The levels of chimeric antibody chain 
synthesis from two producers, D7 and 3E3, are shown in Table 1. Chimeric 
L6 antibody was prepared by culturing the D7 transfectant cells for 24 
hours at 2.times.10.sup.6 cells/ml in 5 l DMEM supplemented with HEPES 
buffer and penicillin and streptomycin. The supernatant was concentrated 
over an Areicon YM30 membrane in 10ram sodium phosphate buffer, pH 8.0. 
The preparation was loaded over a DEAE-Cellulose column, which separated 
the immunoglobulin into unbound and bound fractions. Samples from the 
DEAE-unbound, DEAE-bound and the pre--DEAE preparations (from 1.6 l of 
medium) was separately purified by affinity chromatography on a Protein-A 
Sepharose column, eluting with 0.1M sodium citrate pH 3.5. The eluted 
antibody was neutralized and concentrated by Amicon centricon filtration, 
in phosphate-buffered saline. The yields for the three preparations were 
12 ug (DEAE unbound), 6 ug (DEAE bound), and 9 ug (pre-DEAE column) . 
Western analysis of the antibody chains indicated that they were combined 
in an H 2L.sub.2 tetramer like native immunoglobulins. 
(7) Purification of Chimeric L6 Antibody Secreted in Tissue Culture 
a. Sp2/0.pING2114.D7 cells were grown in culture medium [DMEM (Gibco 
#320-1965), supplemented with 10% Fetal Bovine Serum (Hyclone #A-1111-D), 
10 mM HEPES, 1.times. Glutamine-Pen-Strep (Irvine Scientific #9316) to 
1.times.10.sup.6 cell/ml. 
b. The cells were then centrifuged at 400xg and resuspended in serum-free 
culture medium at 2.times.10.sup.6 cell/ml for 18-24 hr. 
c. The medium was centrifuged at 4000 RPM in a JS-4.2 rotor (3000xg) for 15 
min. 
d. 1.6 liter of supernatant was then filtered through a 0.45 micron filter 
and then concentrated over a YM30 (Amicon Corp.) filter to 25 ml. 
e. The conductance of the concentrated supernatant was adjusted to 5.7-5.6 
mS/cm CDM 80 radiometer and the pH was adjusted to 8.0. 
f. The supernatant was centrifuged at 2000xg, 5 min., and then loaded onto 
a 40 ml DEAE column, which was preequilibrated with 10 mM sodium 
phosphate, pH 8.0. 
g. The flow through fraction was collected and loaded onto a 1 ml protein 
A-Sepharose (Sigma) column preequilibrated with 10 mM sodium phosphate, pH 
8.0. 
h. The column was washed first with 6 ml 10 mM sodium phosphate buffer pH 
=8.0, followed by 8 ml 0.1M sodium citrate pH =3.5, then by 6 ml 0.1M 
citric acid (pH 2.2). Fractions of 0.5 ml were collected in tubes 
containing 50 ul 2M Tris base (Sigma). 
i. The bulk of the IgG was in the pH 3.5 elution and was pooled and 
concentrated over Centricon 30 (Amicon Corp.) to approximately 0.06 ml. 
j. The buffer was changed to PBS (10 mM sodium phosphate pH =7.4, 0.15M 
NaCl) in Centricon 30 by repeated diluting with PBS and reconcentrating. 
k. The IgG solution was then adjusted to 0.10 ml and bovine serum albumin 
(Fraction V, U.S. Bio-chemicals) was added to 1.0% as a stabilizing 
reagent. 
(8) Production in and Purification of Chimeric L6 Antibody from Ascites 
Fluid 
a. The ascites was first centrifuged at 2,000 xg for 10 min. 
b. The conductance of the supernatant was adjusted to 5.7-5.6 mS/cm and its 
pH adjusted to 8.0. 
c. Supernatant was then loaded onto a 40 ml DEAE-cellulose column 
pre-equilibrated with 10 mM Na.sub.2 PO.sub.4 H pH 8.0. 
d. The flow through from the DEAE column was collected and its pH was 
adjusted to 7.4, and the loaded onto a 1.0 ml goat anti-human IgG (H+L) 
-sepharose column. 
e. The column was washed first with 6 ml of 10 mM sodium phosphate, 0.5M 
sodium chloride, followed by 8 ml of 0.5M NH.sub.4 OH, and 3M sodium 
thiocyanate. 
f. The sodium thiocyanate eluate was pooled and dialyzed against 21 PBS 
overnight. 
The antibody can be further concentrated by steps j. and k. of the previous 
procedure. 
TABLE 1 
______________________________________ 
Levels of Secreted Chimeric L6 
Chains from Sp2/0 Transfectants.sup.a 
Sp2/0.D7 Sp2/0.3E3 
Culture Condition 
FBS Kappa.sup.b 
Gamma.sup.c 
Kappa.sup.b 
Gamma.sup.c 
______________________________________ 
1. 20 ml, 2d, + 17 77 100 700 
seed @ 
2 .times. 10.sup.5 /ml 
2. 200 ml, 2d, + 0.9 6 80 215 
seed @ 
2.5 .times. 10.sup.5 /ml 
3. 200 ml, 1d, - 1.9 3.8 97 221 
seed @ 
2 .times. 10.sup.6 /ml 
4. Balb/c ascites 
- 5,160 19,170 ND ND 
______________________________________ 
.sup.a Sp2/0 cells transfected by electroporation with pING2114(pL6HL) 
.sup.b ug/l measured by ELISA specific for human Kappa human BenceJones 
protein standard. 
.sup.c ug/l measured by ELISA specific for human gamma human IgG 
standard. 
ND Not determined. 
FBS: Fetal Bovine Serum 
(9) Studies Performed on the Chimeric L6 Anti-body 
First, the samples were tested with a binding assay, in which cells of both 
an L6 antigen-positive and an L6 antigen-negative cell line were incubated 
with standard mouse monoclonal antibody L6, chimeric L6 antibody derived 
from the cell culture supernatants, and chimeric L6 antibody derived from 
ascites (as previously described) followed by a second reagent, 
fluorescein-isothiocyanate (FITC) -conjugated goat antibodies to human (or 
mouse, for the standard) immunoglobulin. 
Since the binding assay showed strong reactivity of the chimeric L6 on the 
L6 antigen positive cell line and total lack of reactivity on the negative 
cell line, the next step was to test for the ability of the chimeric L6 to 
inhibit the binding of mouse L6 to antigen positive cells; such inhibition 
assays are used routinely to establish the identity of two antibodies' 
recognition of antigen. These data are discussed below ("Inhibition of 
binding"). As part of these studies, a rough estimate of antibody avidity 
was made. 
Finally, two aspects of antibody function were studied, the ability to 
mediate ADCC in the presence of human peripheral blood leukocytes, and the 
ability to kill L6 positive tumor cells in the presence of human serum as 
a source of complement (see "Functional Assays" below) . 
Binding Assays. Cells from a human colon carcinoma line, 3347, which had 
been previously shown to express approximately 5.times.10.sup.5 molecules 
of the L6 antigen at the cell surface, were used as targets. Cells from 
the T cell line HSB2 was used as a negative control, since they, according 
to previous testing, do not express detectable amounts of the L6 antigen. 
The target cells were first incubated for 30 min at 4.degree. C. with 
either the chimeric L6 or with mouse L6 standard, which had been purified 
from mouse ascites. This was followed by incubation with a second, 
FITC-labelled, reagent, which for the chimeric antibody was goat- 
anti-human immunoglobulin, obtained from TAGO (Burlingame, CA), and used 
at a dilution of 1:50. For the mouse standard, it was goat-anti-mouse 
immunoglobulin, also obtained from TAGO and used at a dilution of 1:50. 
Antibody binding to the cell surface was determined using a Coulter Model 
EPIC-C cell sorter. 
As shown in Table 2 and Table 2A, both the chimeric and the mouse standard 
L6 bound significantly, and to approximately the same extent, to the L6 
positive 3347 line. They did not bind above background to the L6 negative 
HSB2 line. 
In view of the fact that the three different chimeric L6 samples presented 
in Table 2 behaved similarly in the binding assays, they were pooled for 
the inhibition studies presented below. The same inhibition studies were 
performed for chimeric L6 derived from ascites fluid presented in Table 
2A. 
Inhibition of Binding. As the next step was studied the extent to which 
graded doses of the chimeric L6 antibody, or the standard mouse L6, could 
inhibit the binding of an FITC-labelled mouse L6 to the surface of antigen 
positive 3347 colon carcinoma cells. 
Both the chimeric and mouse standard L6 inhibited the binding of the 
directly labelled L6 antibody, with the binding curves being parallel. The 
chimeric antibody was slightly less effective than the standard, as 
indicated by the results which showed that 3.4 ug/ml of the pooled 
chimeric L6MAb, as compared to 2.0 ug/ml of the standard mouse L6MAb was 
needed for 50% inhibition of the binding, and that 5.5 ug/ml of the 
chimeric L6 (derived from ascites) as compared to 2.7 Ug/ml of the 
standard mouse L6MAb was needed for 50% inhibition of binding. 
As part of these studies, a rough estimate was made of antibody avidity. 
The avidity of the standard mouse L6 had been previously determined to be 
approximately 4.times.10.sup.8 The data indicated that there were no 
significant differences in avidity between the chimeric and the mouse L6. 
Functional Assays. A comparison was made between the ability of the 
chimeric L6 and standard mouse L6 to lyse L6 antigen positive cells in the 
presence of human peripheral blood leukocytes as a source of effector 
cells (mediating Antibody Dependent Cellular Cytotoxcity, ADCC) or human 
serum as a source of complement (mediating Complement-Dependent Cytolysis, 
CDC). 
As shown in Table 3 and Tables 3A-3D, the chimeric L6 was superior to the 
simultaneously tested sample of mouse L6 in causing ADCC, as measured by a 
4 hr .sup.51 Cr release test. 
Tables 4 and 4A-4B present the data from studies on complement-mediated 
target cell lysis. In this case, a high cytolytic activity was observed 
with both the mouse and the chimeric L6 antibodies. 
CONCLUSIONS 
The results presented above demonstrate a number of important unexpected 
qualities of the chimeric L6 monoclonal antibody of the invention. 
Firstly, the chimeric L6 antibody binds to L6 antigen positive tumor cells 
to approximately the same extent as the mouse L6 standard and with 
approximately the same avidity. This is significant for the following 
reasons: the L6 antibody defines (a) a surface carbohydrate antigen, and 
(b) a protein antigen of about 20,000 daltons, each of which is 
characteristic of non-small cell lung carcinoma (NSCLC) and certain other 
human carcinomas. Significantly, the L6 antibody does not bind detectably 
to normal cells such as fibroblasts, endothelial cells, or epithelial 
cells in the major organs. Thus the chimeric L6 monoclonal antibody 
defines an antigen that is specific for carcinoma cells and not normal 
cells. 
In addition to the ability of the chimeric L6 monoclonal antibodies of the 
present invention to bind specifically to malignant cells and localize 
tumors, the chimeric L6 exerts profound biological effects upon binding to 
its target, which make the chimeric antibody a prime candidate for tumor 
immunotherapy. The results presented herein demonstrate that chimeric L6 
is capable of binding to tumor cells and upon binding kills the tumor 
cells, either by ADCC or CDC. Such tumor killing activity was demonstrated 
using concentrations of chimeric L6 antibody as low as 0.01 ug/ml (10 
ng/ml). 
Although the prospect of attempting tumor therapy using monoclonal 
antibodies is attractive, with some partial tumor regressions being 
reported, to date such monoclonal antibody therapy has been met with 
limited success (Houghton, February 1985, Proc. Natl. Acad. Sci. 
82:1242-1246) The therapeutic efficacy of mouse monoclonal antibodies 
(which are the ones that have been tried so far) appears to be too low for 
most practical purposes. The discovery of the profound biological activity 
of chimeric L6 coupled with its specificity for a carcinoma antigen makes 
the chimeric L6 antibody a choice therapeutic agent for the treatment of 
tumors in vivo. Moreover, because of the "human" properties which will 
make the chimeric L6 monoclonal antibodies more resistant to clearance in 
vivo, the chimeric L6 monoclonal antibodies will be advantageously used 
not only for therapy with unmodifled chimeric antibodies, but also for 
development of various immunoconjugates with drugs, toxins, 
immunomodulators, isotopes, etc., as well as for diagnostic purposes such 
as in vivo imaging of tumors using appropriately labelled chimeric L6 
antibodies. Such immunoconjugation techniques are known to those skilled 
in the art and can be used to modify the chimeric L6 antibody molecules of 
the present invention. 
Two illustrative cell lines secreting chimeric L6 antibody were deposited 
prior to the U.S. filing date at the ATCC, Rockville Maryland. These are 
transfected hybridoma C255 (corresponds to 3E3 cells upra) ATCC HB 9240 
and transfected hybridoma C256 (D7 cells supra) ATCC HB 9241. 
(10) Expression in Yeast of L6 Chains 
Genetic sequences coding for Chimeric L6 antibody heavy and light chains 
were prepared and introduced into vectors. Yeast cells were transformed 
therewith and expression of separate heavy and light antibody chains for 
L6 antibody was detected. 
The present invention is not to be limited in scope by the cell lines 
deposited since the deposited embodiment is intended as a single 
illustration of one aspect of the invention and all cell lines which are 
functionally equivalent are within the scope of the invention. Indeed, 
various modifications of the invention in addition to those shown in the 
art from the foregoing description and accompanying drawings. Such 
modifications are intended to fall within the scope of the appended 
claims. 
TABLE 2 
______________________________________ 
Binding Assays Of Chimeric L6 Antibody and Mouse L6 
Monoclonal Antibody on an L6 Antigen Positive and L6 Antigen 
Negative Cell Line. 
Antibody Batch GAM GAH 
______________________________________ 
Binding Ratio For* 
H3347 Cells (L6 +) 
Standard L6 56.6 4.2 
Chimeric L6 
a 1.3 110.3 
b 1.3 110.3 
c 1.3 110.3 
Binding Ratio For* 
HSB-2 Cells (L6 -) 
Standard L6 1.1 1.1 
Chimeric L6 
a 1.0 1.0 
b 1.0 1.1 
c 1.0 1.1 
______________________________________ 
*All assays were conducted using an antibody concentration of 10 ug/ml. 
The binding ratio is the number of times brighter a test sample is than a 
control sample treated with GAM (FITC conjugated goatanti-mouse) or GAH 
(FITC conjugated goat antihuman) alone. A ratio of 1 means that the test 
sample is just as bright as the control; a ratio of 2 means the test 
sample is twice as bright as the control, etc. 
TABLE 2A 
______________________________________ 
Binding Assays Of Chimeric L6 Antibody and Mouse 
Monoclonal Antibody on an L6 Antigen Positive and L6 Antigen 
Negative Cell Line. 
Antibody 
Concentration 
Antibody (ug/ml) GAM GAH 
______________________________________ 
Binding Ratio For* 
H3347 Cells (L6 +) 
Standard L6 30 38 4 
10 49 4 
3 40 3 
Chimeric L6 30 2 108 
(Ascites) 10 2 84 
3 1 42 
Chimeric L6 30 1 105 
(Cell Culture) 
10 1 86 
3 1 44 
Binding Ratio For** 
HSB-2 Cells (L6 -) 
Standard L6 10 1 1 
Chimeric L6 10 1 1 
(Ascites) 
Chimeric L6 10 1 1 
(Cell Culture) 
______________________________________ 
*The binding ratio is the number of times brighter a test sample is than 
control sample treated with GAM (FITC conjugated goat antihuman) alone. A 
ratio of 1 means that the test sample is just as bright as the control; a 
ratio of 2 means the test sample is twice as bright as the control, etc. 
TABLE 3 
______________________________________ 
ADCC of Chimeric L6 (Mouse) L6 Antibodies On Colon 
Carcinoma Cell Line C3347. 
Antibody 
Concentration 
PBL per % 
Antibody (ug/ml) Target Cell 
Cytolysis* 
______________________________________ 
Chimeric L6 
10 100 64 
(Cell Culture) 
5 100 70 
10 0 2 
Standard L6 
10 100 24 
5 100 17 
10 0 2 
None 0 100 1 
______________________________________ 
*The target cells had been labelled with .sup.51 Cr and were exposed for 
hours to a combination of MAb and human peripheral blood leukocytes (PBL) 
and the release of .sup.51 Cr was measured subsequently. The release of 
.sup.51 Cr (after corrections of values for spontaneous release from 
untreated cells) is a measure of the percent cytolsis. 
TABLE 3A 
______________________________________ 
ADCC of Chimeric L6 and Standard (Mouse) L6 Antibodies On 
Colon Carcinoma Cell Line C3347. 
Antibody 
Concentration 
PBL per % 
Antibody (ug/ml) Target Cell 
Cytolysis* 
______________________________________ 
Chimeric L6 
20 100 80 
(Ascites) 10 100 74 
5 100 71 
2.5 100 71 
20 0 0 
Chimeric L6 
10 100 84 
(Cell Culture) 
5 100 74 
2.5 100 67 
10 0 3 
Standard L6 
20 100 32 
10 100 26 
20 0 0 
______________________________________ 
*The target cells had been labelled with .sup.51 Cr and were exposed for 
hours to a combination of MAb and human peripheral blood leukocytes (PBL) 
and the release of .sup.51 Cr was measured subsequently. The release of 
.sup.51 Cr (after corrections of values for spontaneous release from 
untreated cells) is a measure of the percent cytolsis. 
TABLE 3B 
______________________________________ 
ADCC of Chimeric L6 and Standard (Mouse) L6 Antibodies On 
Colon Carcinoma Cell Line C3347. 
Antibody 
Concentration 
PBL per % 
Antibody (ug/ml) Target Cell 
Cytolysis* 
______________________________________ 
Chimeric L6 
5 100 84 
(Ascites) 2.5 100 78 
1.25 100 85 
0.63 100 81 
0.31 100 80 
0.16 100 71 
0.08 100 65 
5 0 0 
Standard L6 
5 100 32 
5 0 0 
None 0 100 19 
______________________________________ 
*The target cells had been labelled with .sup.51 Cr and were exposed for 
hours to a combination of MAb and human peripheral blood leukocytes (PBL) 
and the release of .sup.51 Cr was measured subsequently. The release of 
.sup.51 Cr (after corrections of values for spontaneous release from 
untreated cells) is a measure of the percent cytolsis. 
TABLE 3C 
______________________________________ 
ADCC of Chimeric L6 and Standard (Mouse) L6 Antibodies On 
Melanoma Cell Line M2669. 
Antibody 
Concentration 
PBL per % 
Antibody (ug/ml) Target Cell 
Cytolysis* 
______________________________________ 
Chimeric L6 
10 100 35 
(Ascites) 1 100 31 
0.1 100 27 
0.01 100 15 
0.001 100 13 
0.0001 0 15 
Standard L6 
10 100 9 
1 100 15 
None 0 100 9 
Chimeric L6 
10 10 19 
(Ascites) 1 10 15 
0.1 10 11 
0.01 10 13 
0.001 10 22 
0.0001 10 11 
Standard L6 
10 10 7 
1 10 6 
None 0 10 8 
Chimeric L6 
10 0 4 
(Ascites) 
Standard L6 
10 0 9 
______________________________________ 
*The target cells had been labelled with .sup.51 Cr and were exposed for 
hours to a combination of MAb and Human peripheral blood leukocytes (PBL) 
and the release of .sup.51 Cr was measured subsequently. The release of 
.sup.51 Cr (after corrections of values for spontaneous release from 
untreated cells) is a measure of the percent cytolysis. 
TABLE 3D 
______________________________________ 
ADCC of Chimeric L6 and Standard (Mouse) L6 Antibodies On 
Colon Carcinoma Cell Line C3347. 
Antibody 
Concentration 
PBL per % 
Antibody (ug/ml) Target Cell 
Cytolysis* 
______________________________________ 
Chimeric L6 
10 100 62 
(Ascites) 1 100 66 
0.1 100 69 
0.01 100 26 
0.001 100 8 
0.0001 0 3 
10 0 0 
Standard L6 
10 100 19 
1 100 24 
0 0 
None 0 100 8 
______________________________________ 
*The target cells had been labelled with .sup.51 Cr and were exposed for 
hours to a combination of MAb and Human peripheral blood leukocytes (PBL) 
and the release of .sup.51 Cr (after corrections of values for spontaneou 
release from untreated cells) is a measure of the percent cytolysis. 
TABLE 4 
______________________________________ 
Complement-dependent cytotoxic effect of chimeric and stan- 
dard (mouse) L6 on colon carcinoma cells from line 3347, as 
measured by a 4-hr .sup.51 Cr-release assay. Human serum from a 
healthy subject was used as the source of complement. 
Antibody Human complement 
% Cytolysis 
______________________________________ 
L6 Standard 10 ug/ml 
Yes 90 
L6 chimeric 10 ug/ml 
Yes 89 
L6 Standard 10 ug/ml 
No 0 
L6 chimeric 10 ug/ml 
No 1 
______________________________________ 
TABLE 4A 
______________________________________ 
Complement Dependent Cytotoxic Effect of Chimeric L6 and 
Standard (Mouse) L6 Antibodies on Colon 
Carcinoma Cell Line C3347 
Antibody 
Concentration 
Human % 
Antibody (ug/ml) Complement Cytolysis* 
______________________________________ 
Chimeric L6 
20 + 29 
(Ascites) 10 + 23 
5 + 18 
2.5 + 8 
20 Inactivated 
0 
10 0 0 
Chimeric L6 
20 + 29 
(Cell Culture)) 
5 + 26 
2.5 + 18 
20 Inactivated 
0 
10 0 4 
Standard L6 
20 + 55 
10 + 37 
20 Inactivated 
0 
20 0 1 
None 0 + 0 
______________________________________ 
*Complement mediated cytolysis was measured by a 4 hour .sup.51 Crrelease 
assay. Human serum from a healthy subject was used as the source of 
complement. 
TABLE 4B 
______________________________________ 
Complement Dependent Cytotoxic Effect of Chimeric L6 and 
Standard (Mouse) L6 Antibodies on Colon 
Carcinoma Cell Line C3347 
Antibody 
Concentration 
Human % 
Antibody (ug/ml) Complement Cytolysis* 
______________________________________ 
Chimeric L6 
10 + 209 
(Ascites) 5 + 155 
2.5 + 166 
1.25 + 114 
0.6 + 63 
0.3 + 17 
10 0 0 
Standard L6 
10 + 96 
5 + 83 
2.5 + 48 
1.25 + 18 
0.6 + 7 
0.3 + 4 
10 0 2 
None 0 + 0 
______________________________________ 
*Complement mediated cytolysis was measured by a 4 hour .sup.51 Crrelease 
assay. Human serum from a healthy subject was used as the source of 
complement.