CA2115346A1 - Chimeric anti-cea antibody - Google Patents

Abstract

A chimeric murine human antibody, the kappa and gamma genes of which have a murine variable region and a human constant region, are described.

Description

WO 93/25237 ,~ 1 i J ~ l~ li PCI'/US93/0~709 , ': .s ~

CHIMERIC ANTI-CEA ANTIBODY
. ~
This invention was made with government support under Grant No. CA 43904 awarded by the National Institutes of Health. The government has certain rights in the invention. ' FIELD OF THE INVENTION
This invention relates to a chimeric mouse-human ~;~
antibody to carcinoembryomic antigen ( CEA) designated ~ ' T84.12. ,~
BACKGROUND OF THE INVENTION ', CEA is a widespread tumor marker. Its~expression ,,~
can be detected in more than 95% of all human colon '~`
cancers. It is a member of the immunoglobulin ~ ' superfamily~and i8 closely related to NCA and BGP. '~'~
~of the various available CEA~specific monoclonal ,,, ant~ibodi~es~ murine T84.66 antibody shows~the highest spec,ificity~and~affinity for CEA (Wagener, et al., J.,~,Immunoloqy 130:2308-2315 (1985)). It has been ,' used'~suocessfully for ln vivo tumor imaging in mice '`
and'humans. ~It is well suited for the immunodetection and immunotherapy of human colon ;' -- ; -~, cancers.
The }n vivo human~use of T84.66 is limlted by its mur'ine origin resulting~in~immune response against '~' the heterologous~immunoglobulin. Chimeric T84.66 was ':, 'areated~by use~of~recombinant gene technology to lessen the immunogenicity in man See Neumaier, et ,,~
` al~ ancer ~esearchl50:2128-2134 (1990)j and United ```-States Patent 5,081,235. The cloned antibody genes '' including the immunoglobulin promoter were i~
trans~ected into SP2/0 myeloma cells by electroporation or CHO cells using lipofection. The expressed chimeric mabs were characterized,in ' I -~
, ' different enzyme immunoassays and a western blot. ', ~; :~: . . ~.' ~ ~ .
~ . ~

W093/25237 ~ ~ ~ J ~ ~'; PCT/US93/0~709 -. .", .

The sequence of the V-regions of the heavy and light chain genes were determined using the well known ~ I
Sanger chain termination method.
SUMMARY OF THE INVENTION
Murine T84.12 is another well characterized CEA
specific monoclonal of the murine IgG2a isotype. It recognizes the same epitope on CEA as T84.66 but with an affinity constant which is lower by a factor of approximately ten (10). For that reason, T84.12 was selected, pursuant to this invention, to generate mouse-human chimeric antibodies for therapeutic -~
purposes in man.
cDNA clones were humanized (chimerized) by shuffling the human IgG1 heavy or light chain ~
constant domain exons, including the S'-UT and leader -peptide, to the variable regions of the heavy and light chain genes of murine T84.12. -;~
The~resulting hybridoma produces significant quantities~ of chimeric T84.12 anti-CEA antibodies - useful for, among other things, human therapeutic ``
purposes.
DETAILED DESCRIPTION OF THE INVENTION -~
Production of the chimeric anti-CEA antibodies of~`
this~invention entails a series of steps including, -;~
among others, identification of the amino terminal ~;
; proteinl sequences of murin- T84.12, determination of the cDNA~sequence of mouse light chain and heavy chain clones of T84.12 and of the corresponding amino ~;~
acid sequences and the chimerization of murine T84.14 -cDNA clones. One aspect of the invention entails ln ~ vi~rolmutagèh~sis of'aim'ouse T84.12 light chain clone.
; Am:inoterminal Sequences of Murine T84.12 Murine T84.12 specific light (L) chain clones Ll-L4 and T84~1~2 heavy chain clones Hl-H4 were prepared and~sequenced in known manner. All four ¦~
heavy chain clones showed a 100% V-region homology in¦`~

:

W093/25~37 ~ PCT/US93/05709 ;. ,;:

their V-region and therefore clone H4 was selected for the sequencing of the IgG2a heavy chain constant regions. The variable domains of light chain clones L2, L3 and L4 were identical. Clone Ll was totally different, apparently representing the endogenous ^~
transcript. For the complete characterization of the ~-constant kappa light chain domains and the 3'-untranslated region the light chain clones Ll, L4 and the heavy chain clone H4 were selected. -~
Table I sets forth the amino terminal sequences ~
o~ the T84.12 light and T84.12 heavy chains. The ~-reported sequences were determined using reduced (DTT) and alkylated (iodoacetic acid) purified monoclonal antibody. The heavy and light chains were ~-separated under reducing conditions on a Sephadex GlOO column using 1 M acetic acid as a running -, buffer. The isolated chains were subjected to amino acid sequencing.

TABLE I
~ Resldue T84.12 liqht T84.12 heavy ~`~
- ~ ~ 1 Asp Glu 2 Ile Val -3 Val Lys 4 Leu Leu ~
Thr Val ~`
6 Gln Glu - 7 Ser Ser ;~;
8 Gln Gly ~ -9 Lys Gly ~
Phe Gly -11 Met Phe 12 Gly Val ~3 Thr Lys 14~ Ser ~ Pro - Gly :: :
- :
;;.,~, ~ , ~::

~",, 3/2~237 ' PCT/US~3/0~709 cDNA_Sequence of Mouse Liqht Chain Clone T84.12 L4 The sequence of full size cDNA T84.12 clone was determined (1020 bp) in known manner. This clone contained a very short 5'-UT region of 10 bp which was followed by the ATG start codon. The presence of the entire leader peptide, V-region and the cKappa constant domain could be demonstrated. At the end of the Ckappa constant domain a TAG stop codon was present. The 3'-untranslated region (2B0 bp) -~
contained a polyadenylation signal (AATAAA~ and a ;
poly(A~ tail. The entire full size cDNA clone was flanked by the destroyed Smal restriction cloning ;,~
site (GGG-CCC). The ~ranslation of the obtained nucleotide sequence into the amino acid sequence ;
yielded an open reading frame (bp 34-741 = 708 bp) resulting in 236 amino acids. In addition the Ckappa ~ ~-constant domain showed a 99.7% homology to other ;
;published Ckappa constant domain sequences (Kabat). ;^`~
There was only a C to T exchange (see Xabat,~et al., "Sequences of Proteins of Immunological Interest", Fourth Ed. U.S. Dept. of Health and Human Services PHS NIH (1987)) in the T84.12 light chain sequence at ~;~
bp 711 (CATTGT). This base pair difference resulted `~
in a silent mutation. The leader peptide and ~-~
.
V-region were different from the T84.66 clones.
In SEQ ID N0. I, the light chain cDNA sequence of murine T84.12, the following regions are underlined (from the top to the bottom): ATG start codon, start of variable region, start of C-kappa constant domain, TAG stop codon and polyadenylation signal. ;~
Aminio Acid!Sequenceio~f T84.12 L4 (Frame 1 = 34-741) In SEQ ID. N0. 2, the light chain amino acid ' sequence of T84.12, the following regions are underlined (from the top to the bcttom): ATG start J
codon, start of variable region, start of C-kappa constant domain and TAG stop codon.

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W093/252~7 A~ PCI`/US93/05709 ~ .

cDNA Sequence of Mouse HeavY Chain Clone T84.12 H4 ; ;~
The complete sequence of the full size cDNA clone T84.12 was determined in known manner (1645 bp).
This clone contained a 10 bp longer 5'-UT region than the light chain clone L4 which was also followed by the ATG start codon. The presence of the entire 'r leader peptide, V-region and all three constant domain of IgG2a could be demonstrated. At the end of CH3 constant domain of IgG2a a TGA stop codon was present. The 3'-untranslated region (120 bp) contained the polyadenylation signal AATAAA. The entire full size cDNA clone wàs flanked by the destroyed Smal restriction cloning site GGG-CCC. The translation of the obtained nucleotide sequence into ~;
the amino acid sequence yielded an open reading frame (52-1485 = 1434 bp) resulting in 478 amino acids. In ;~-addition, the IgG2a constant domain showed a 98.7% ~;
homology to other published~IgG2a constant~domain ~-sequences (Kabat). The hinge region showed a 100% -homology to the Kabat sequence too. Two different codons~in~the CHl domain were identical to IgG3 and ~;
three different codons in the CH3 domain identical to MOPC21. `~-~
In SEQ ID. NO. 3, the heavy chain cDNA sequence of T84.1~, the following regions are underlined (from the top to the bottom~: ATG start codon, start of variable region, start of CHl constant domain, start of hinge region,~;start of CH2 constant domain, start ;-o~ CH3~constant domain, T~A stop codon and polyadenylation signal.
Amino Acid`!'Sequèn;celof T84.12 H4 (Frame 2 = 52-1485) In SEQ ID NO. 4, the heavy chain amino acid -~
~;~ sequence of T84.12 H4, the following regions are underlined (from the top to the bottom): ATG start W~93/2s237 ~ PCT/US93/05709 codon, start of variable region, start of CH1 constant domain, start of hinge region, start of CH2 constant domain, start of CH3 constant domain and TGA
stop codon.
Chimeric T84.12 , The obtained and characterized full size cDNA
murine T84.12 L4 and H4 clones were chimerized using the constant domains of human IgGl heavy chain cDNAs ~;
and the constant domains of human kappa light chain -~
cDNAs respectively. The human heavy and kappa chain constant region sequences were derived from plasmids obtained from Dr. Jeffrey Schlom, National Institutes of Health. The plasmids contained chimeria B72.3 cDNA clones, cloned from cells expressing the ~ -~
chimeric B72.3 antibody (see, Hutzell, et al. t Cancer `~
Research 31:181-189 (1991)~. Dr. Schlom's group obtained the human gamma and kappa chain genomic expression vectors from Dr. Sherie Morrison, UCLA
(oi, V.T., et al., Biotechniques 4:214 (1986)), in order to make those constructs. Using specific primers, the variable domains of T84.12 (mouse cDNA) ;~
were, in known manner, fuæed in frame to the human constant domain(s) of chimeric B72.3 using the splice overlap extension PCR. See Ho, et al., Gene 77:51-59 988) and Horton, et al., Gene 77:61-68 (1989). -~
These full size cDNA's were named CHI T84 . 12 L3, L6, L8, H2 and H3. -~
The chimeric clones were used for the production of Fab, F(ab')2-fragments, Fv-fragments and of single chain antibodies linked by a synthetic peptide.
~ dDNA'Seguence of T84.I2 L6 The entire sequence of the full size cDNA clone ~ ~.
chiT84.12 L6 was determined in known manner (956 bp). The clone chiT84.12 L6 showed the correct , ~ .
'' '~

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W O 93t25237 ~ P ~ /US93/05709 , ....
sequence for a mouse-human chimeric T84.12 light ;~-chain. The clone chiT84.12 L6 was used for further subcloning into the pH~-Apr-neo vector (see Gunning, et al., Proc. Natl. Acad. Sci. 84:4831-4835 ~1987)) to transfect SP2/0 myeloma cells. ~;
- The clone chiT84.12 L6 contained a short 5'-UT ;;
region of 9 bp which was followed by the ATG start codon. The presence of the entire leader peptide, . ~, i V-region and the human Ckappa constant domain could be confirmed. At the end of the human Ckappa ~-; constant domain a TAG stop codon was present. The 3'-untranslated regi~n (218 bp) contained a `~`
polyadenylation signal (AATAAA). The translation of ~
the obtained nucleotide seque~nce into the amino acid ~ ``
sequence yielded an open reading frame (bp 34-738 = `
705 bp~ resulting in~235 amino acids. In addition ~ -;
the~human ~CXappa con~stant domain showed a 100 homology~to~other~published Ckappa constant domain sequences~(Ka~:at).
~ In~SEQ~ID~NO.~7,~the 1ight chain cDNA sequence of ;~
7~ chiT84~12~L6,~ the~following~r~egions are underlined (from-the~top~to thè bottom): ATG start codon, start ``i;~
of~ ~ se~variable~region, start of human C-kappa ii~
constant~domain, TAG stop~codon and polyadenylation Codinq~Sequence~of ChiT84.12~L6 (bp = 34-738) ~--In~Seq~ ID~No;.~ 8,~the~1iqht chain amino acid ` -sequence of: chiT84~.~12~L6~, the~following regions are `:~:
undérlined ~(~from -the top to the bottom): ATG start codon, start of~mouse variable region, start of human C-kappa c onst~antldomaiin!and TAGi~stop coqon.

, , . - . ~ .: . .

W093/2s237~ PCT/US93/05709 ~': ' .' cDNA Sequence of Chimeric T84.12 H3 ;~
The complete sequence of the full size cDNA clone chiT84.12 H3 was determined in known manner (1641 bp). The clone chiT84.12 H3 showed the correct sequence for a mouse-human chimeric T84.12 heavy chain and had one mutation at the beginning of the CH2 domain (GTG to GCG at position 484 = valine against alanine) and one at the end of the 3'-UT
(AAATAAA to GAATAAA). However, this did not affect ;~
the polyadenylation signal. The clone chiT84.12 H3 was used for further subcloning into the pH~-Apr-gpt --~
vector to transfect SP2/0 myeloma cells which are expressing chiT84.12 kappa light chains.
This clone contained a 41 bp long 5'-UT region which was followed by the ATG start codon. The presence of the entire leader peptide, mouse V-region and all three human constant domain of IgGl could be demonstrated. At the end of CH3 constant domain of IgG1 a TGA stop codon was present. The 3'-untranslated region (153 bp) contained the ~-polyadenylation signal AATAAA. The translation of ~-~
the obtained nucleotide sequence into the amino acid ~;~
sequence yielded an open reading frame (bp 52-1485 =
1410 bp) resulting in 470 amino acids. In addition, the human IgGl constant domain showed a 100% homology to other published IgGl constant domain sequences (Kabat). The hinge region showed a 100~ homology to the~Kabat sequence too.
In SEQ ID N0.~9, the heavy chain cDNA seguence of chiT84.12 H3, the following regions are underlined (from'the top tolthelbottom): ATG starticodon, start of mouse variable region, start of human CHl constant domain, start of hinge region, start of CH2 constant .~``
domain, start of CH3 constant domain, TGA stop codon and polyadenylation signal. ;~
,; ~
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'~`, W O 93/25237 PC~r/US93/~5709 ~-:
~11JC)4Ij _9_ ::

In SEQ ID NO. 10, the heavy chain amino acid sequence of chiT84.12 H3, the following regions are underlined (fro~ the,top to the bottom): ATG staxt ';~'-codon, start of mouse variable region, start of human , ~
CHl constant domain, start of hinge region, start of ~, CH2 constant domain, start of CH3 constant domain and TGA stop codon. ,~
In Vitro Mutaqenesis of Mouse T84.12 L4 cDNA ,--With some exceptions, two cysteine residues are typically present in an immunoglobulin domain. The ,'~, CDR3 (L3) of T84.12 light chain clone L4 contained an additional third cysteine residue in the mouse variable kappa light chain domain. The presence of ~, the third cysteine is apparently related to the loss , of binding activity by murine T84.12 after dissociation of both chains and chemical crosslinking -`' - using homobifunctional crosslinking agents. '',' Therefore the cysteine (TGT) in position 364-366, see ,~
SEQ ID'NO. l, (amino acid residue 91) was changed to ;~
a serine (TCT) by site directed mutagenesis. ~-~
Overview of MUTA-GENE Phaqemid In Vitro Mutaqenesis ~-' :~ ~ The mutagenesis was carried out using the '~
MUTA-GENE phagemid in vitro mutagenesis kit from ~,~
BioRad. The original procedure was simplified and ~
- reduced to the following eleven steps: ~, 1. Subcloning of the coding cDNA strand in pTZ18U or pTZ19U phagemids (depending on the orientation of cloned cDNA in pUC18). ~-- 2. Electrotransformation of E. coli CJ236 with ~`
pTZ18U or l9U containing the cDNA to be mutagenized ~:
(plate' on LB'a~pl+ 30''~g/ml chloramphenicol).
3. Miniprep DNA isolation from single - recombinant CJ236 colonies. This E. coli strain '~
incorporates uracil residues into the phagemid DNA.

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W093/25237 ,~ PCT/US93/05709 4. Growth of uracil containing phagemids in 2xYT
media (containing ampicillin (50 ~g/ml) and ~;~
chloramphenicol (30 ~g/ml). Start out with a marked and mini prep DNA analyzed single colony from the plate. Add the helper phage M13K07 in order to~
obtain single stranded phagemid DNA.
5. PEG extraction and purification (PCI) of ~
single stranded phagemid DNA. -6. Phosphorylation of the mutagenesis primer (represents the minus strand and binds to the single -~
stranded plus strand phagemid DNA).
7. Synthesis of the mutagenic strand by annealing of the phosphorylated mutagenesis primer to the purified single stranded phagemid DNA. The ~
complementary minus strand is created by the T4 DNA -~;
polymerase and gaps sealed with the T4DNA ligase. ;~
8. Electrotransformation of E. coli MV1190 with double-stranded mutagenized cDNA. This strain ;~
removes uracil residues. `~
; 9. Isolation af miniprep DNA from single growing re~co~ inant MVllgO colonies. The insert size can be ~determined~by restriction enzyme digest and compared ;~
to the wild type. i~
10. Sequence several miniprep DNA from the mutants and compare it with the wild type sequence.
Select clones with the correct mutations and grow a larger culture (lO0 ml). Purify the mutagenized aDNA using Qiagen columns and confirm the -~
entire sequence of the mutated cDNA clone. ~`
- One such clone, named T84.12 L4-12-1 was selected for exemplification of the invention. `

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W093/2~237 1 J ~ 4 ~ PCT/US93/05709 ., ,- ,, . -cDNA Sequence of T84.12 L4-12~
The entire sequence of the full size cDNA clone ' T84.12 L4-12-1 was determined in known manner (1999 bp). The clone showed the correct sequence for a ,~
mouse T84.12 light chain and the introduced cysteine to serine mutation. It was used for further subcloning into the PH~-Apr-neo vector (See Gunning, et al., Proc. Natl. Acad. Sci. 84:4831-4835 (1987)) `~
to transfect SP2/0 myeloma cells.
This T84.12 L4-12-1 clone contained a very short ,,~, 5'-UT region of 10 bp which was followed by the ATG ~ .' start codon. The presence of the entire leader - ,, peptide, V-region and the Ckappa constant domain ''' could be demonstrated. At the end of the Ckappa constant domain a TAG stop codon was present. The 3'-untranslated region (280 bp) contained a ~
polyadenylation signal (AATAAA). The entire full ~, size cDNA clone was flanked by the destroyed Smal ~' restriction cloning site (GGG-CCC). The translation `~
of the obtained nucleotide seguence into the amino ~acid sequence yielded an open rcading frame (bp -,~ 34-74`1 = 708 bp) resulting in 236 amino acids. In ` ~
addition, the Ckappa constant domain showed a 99.7~ ~''`-~: .
homology to other published cKappa constant domain ,~
sequences (Rabat~. Therè was only a C (Kabat) to T
exchange in the T84.12 light chain sequence at bp 711 (CATTGT). This base pair difference resulted in a ;,~
silent mutation. '-'"
In SEQ ID NO. 5, the light chain cDNA sequence of T84.12 L4-12-1, the following regions were underlined ,'~' (from the,top to the bottom): ATG start codon, start '~
of mouse variable region,'start of human C-kappa ' '`, constant domain, TAG stop codon and polyadenylation signa}. The mutagenized TGT (cys) to TCT (ser) is ' underlined and in italics. '~
. '`'' , ~ ~
~ . .

- ~

W093/25~3~ .ili~J~'~ b PCT/US93/05709 ;~

Amino Acid Sequence of T84.12 L4-12-1 (Frame 1 = 34-741) In SEQ ID NO~ 6, the light chain amino acid sequence of T84.12, the following regions are - underlined (from the top to the bottom): ATG start codon, start of variable region, start of C-kappa constant domain and TAG stop codon. The mutagenized ;
TGT (cys) to TCT (ser) is underlined and in italics.
All other cysteine residues are underlined.
ExPression of Mutaqenized Mouse T84.12 cDNAs The mutated light chain (T84.12 L4-1) cDNA and ~-the normal heavy chain (T84.12 H4) cDNA were transferred in a ~-actin cDNA expression vector (Gunning, et al., supra) and cotransformed into Sp2/0 myeloma cells by electroporation. The vectors include the human ~-actin promoter, ~ntervening `~
sequence, cloning site, and a polyadenylation ~i;
signal. Since the vectors contain the neomycin-res~istance gene, transfectants were selected -~
in~the~presence of the drug, G418. Clones were ~` expanded~and evaluated for antibody production (kappa or gamma chain) and CEA-binding activity by ELISAs. ~``
Although levels of expression were low, therè was a correlation between antibody and anti-CEA activity in `~;~
culture supernatants. ~ i Bindlnq~Activity of T84.12 cYs --> Ser Mutant ~ `~
Kappa chain Gamma chain Anti-CEA activity CIone ~ (nq/ml) (nq/ml)~ (n~ml) , 4Cl 2-6 2-6 2 4H9 6-18 6-18 3-8 `-``-lBl 66_18 22_6 1-3 ``
- ~~5A11 ~!~2l-6~ , 2-6 1-3 ~,: ~
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W~3t2~237 .,1 i J ~ 4 ~ PC~/US93/0~709 SEQUENCE LISTING :
(1) GENERAL INFO~MATION: :
(i) APPLICANT: John E. Shively Rainer Fischer - Anna Wu Ray Paxton ~
Y.H. Joy Yang :~ ;
(ii) TITLE OF INVENTION: Chimeric Anti-C~EA
Antibody (iii) NUMBER OF SEQUENCES: 10 (iv) CO~RESPONDEMCE ADD~ESS:
(A) ADDRESSEE: City of Hope (B) STREET: 1500 East Duarte Road ~.
(C) CITY: Duarte (D) STATE: California (E) COUNTRY: United States of America .. .~ .
(F)~ ZIP: 91010~-0269 ~
.
(v) COMPUTER READABLE FORM:
: : (A) MEDIUM TYPE: 3M Double Density 5 -`~-1/4'l diskette -`~
- (~) COMPUTER: Wang PC -` "
(C) OPERATING~SYSTEM: MS-DOS (R) Version .30 (D) SOFTWARE: Microsoft (R) ~
: (vi~ ~CURRENT APPLICATION DATA: `-(A) APPLICATION NUMBER: 07/904,074 `
(B) FILING DATE: 15 June 1992 ~:`
(C) CLASSIFICATION: Unknown (vii) PRIOR APPLICATION DATA: None .

W~) 93/25~37 r~ j PCriUS93/05709 # ~
-14- ;
(viii) ATTORNEY/AGENT INFORMATION~
(A) NAME: Irons, Edward S. `~
(B) REGISTRATION NUMBER: 16,541 :~
(C) R~FERENCE/DOCKET NUMBER: None ~ .
.::
(ix) TELECOMMUNICATION INFORMATION~
(A) TELEPHONE: ~202) 785-6938 .`'-(B) TELEFAX: (202) 785-5351 :
(C) TELEX: 440087 LM WSH ~
-,:
(2) INFORMATION FOR SEQ ID NO: 1: ;
(i) SEQUENCE CHARACTERISTICS: .
(A) LENGTH: 1041 .
..~ , . .
(B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single Stranded :(D) TOPOLOGY: ~Unknown MoLECULE TYPE: ~Nucleic ACid NYPOTHETICAL: Not Appl~icable ~i -(iv) ANTI-SENSE: Not Applicable /~
:- ~......
(v) FRAGMENT TYPE: Not Applicable~
(Vi) ORIGINAL SOURCE: Synthically Prepared `, (vii) IMMEDIATE SOURCE: Synthetically Prepared (viii) POSITION IN GENOME: None "-~
( ix ) FEATURE: None (x) PUBLICATION INFORMATION: None txi) SÉQUENCEiDESCRIPTION: SEQ ID NO: 1: ! ~ :.:
j,' ~''.

:~ , " :' :-:"
' ' ' WO 93/2~237 ,~ PCT/US~3/0~709 .; :.............................................................................. :
-15- ~.

TGCTGACCCA GTCTCAAAAA TTCATGTCCA CATCAGTTGG AGGCACGGTC 15n GATTTCACTC TCACCATCAG CAATGTGCAG TCTGAAGACT TGGCAG~ATA 350 GTATGAACGA CATAACAGCT ATACCTGTGA GGCCACTCAC AAGACATCAA 700 :~:

CATGCTAATA TTTGCAGAAA ATATTCAATA AAGTGAGTCT TTGCACTTGA 950 -:.
~ ~ AAAA~ 1000 AAAAAAAAAA AAGGGGATCC TCTAGAGTCG ACCTGCAGGC A1041 , -:
(2) INFORMATION FOR SEQ ID NO: 2: .
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 235 -. - .~
(B) TYPE: Amino Acid .;
(C) STRANDEDNESS: Single Stranded ;
(D) TOPOLOGY: Unknown - -(ii) MOLECULE TYPE: Amino Acid ~.
(iii) HYPOTHETICAL: Not Applicable :~
(iv) ANTI-SENSE: Not Applicable . ~:
(v) FRAGMENT TYPE: Not Applicable :
(vi) ORIGINAL SOURCE: Synthetically Prepared ~1;
(vii) IMMEDIATE SOURCE: Syntehtically Prepared :~:
(viii) POSITION IN GENOME: None ,.:
(ix) FEATURE: None ¦ -(x) PUBLICATION INFORMATION: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: . ~.
.~ .

W093/2sZ37 ~ PCT/US93tO5709 Met Glu Ser Gln Thr Gln Val Phe Val Tyr Met Leu Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Leu Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly Gly Thr Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val His Thr Asn Val Ala Trp Tyr Gln Gl~ Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr - . Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp . 85 90 Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Cys Asn Ser Tyr Pro Leu Phe Thr Phe Gly Ser Gly Thr Thr Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly : }40 145 150 Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val i 170 175 180 Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser : ~ 185 190 195 MET~Ser:Ser Thr~Leu Thr Leu Thr Lys:Asp Glu Tyr Glu Arg His 200 : 205 210 Asn:~Ser~Tyr~Thr~Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile;Val Lys Ser Phe Asn Arg Asn GIu Cy5 : 230 : 235 ~
, . ~
(2~INFORNATION FOR SEQ ID NO: 3: ~;
(i) SEQUENCE C~ARACTERISTICS: .;.
, ~ ~ , " : , : .
(A) LENGTH: 1645 (B) TYPE: Nucleic Acid ~`-~;: :, : : . , !
(C~STRANDEDNESS: Single Stranded (D) TOPOL0GY:~ Unknown :.
; (ii) MOLE¢ULE TYPE:~Nucleic Acid : : (iii) HYPOTHETICAL: Not Applicable (iv) ANTI-5ENSE:: Not Applicable , .
~ : (v) FRAGMENT TYPE: Not Applicable :::
~ (vi) ORIGINAL SOURCE: Synthetically Prepared ~' '~ .

:

W093/25237 ~ 1 1 J ~ PCT/US93/05709 ,. .. . . .

(vii) IMMEDIATE SOURCE: Synthetically Prepared (viii) POSITION IN GENOME: None .:~
(ix) FEATURE: None (x) PUBLICATION INFORMATION: None ~ r (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ' TTACGAATTC GAGCTCGGTA CCCCCTGGAT TTGAGTTCCT CACATTCAGT 50 . `
CATGAGCACT GAACACAGAC ACCTCACCAT GAACTTCGGG TTCAGCCTGA 100 '"~
TTTTCCTTGT CCTTGTTTTA AAAGGTGTCC:AGTGTGAAGT GAAGCTGGTG 15Q .:,,'' GAGTCTGGGG GAGGcTTTGT GAAGCCTGGA GGGTCCCTGA AACTCTCCTG 200 .

AGACTCCAGA GAAGAGGCTG GAGTGGGTCG CATCCATTAG TAGTGATGGT 300 ~.:

: ACACGGCCAT GTATTACTGT GCAAGAATCG ACTACTACGG AGGAGGGGGA 450 .':.,;
TTTGGTTACT GGGGC,CAAGG GACTCTGGCC ACTGTCTCTG CAGCCAAAAC 500 . ' AR.CAGCCCCA TCGGTCTATC CAC$GGCCCC TGTGTGTGGA GATACAACTG SSO ~.,.`'.
GCTCCTCGGT GACTCTAGGA TGCCTGGTCA AGGGTTATTT CCCTGAGCCA 600 .'.. ,'~
GTGACCTTGA CCTGGAACTC TGGATCCCTG TCCAGTGGTG TGCACACCTT 650 -~
CCCAGCTGTC CTGCAGTCTG ACCTCTACAC CCTCAGCAGC TCAGTGACT,G 700 TAACCTCGAG CACCTGGCCC AGCCAGTCCA TCACCTGCAA TGTGGCCC'AC 750 .~<',' CCGG~ ~ GCA:GCACCAAGGT GGACAAGAAA ATTGAGCCCA GAGGGCCCAC 800 ~',~''~
AAT ~ CCC~TGTCTCCAT:GCAAATGCCC~AGCACCTAAC CTCTTGGGTG 850 ''~
GACCATCCGT~CTTCATGTTC CCTCCAAAGA TCAAGGATGT ACTCATGATC 900 ~.~'-.',' : ~TC:CTGAGCC~CCATAGTCAC ATGTGTGGTG:~GTGGATGTGA GCGAGGATGA 950 ,.,'.
; ~CCCAGATGTC CAGAT~CAGCT~GGTTTGTGAA CAACGTGGAA GTACACACAG 1000 ~.,.'-,.
:.: :CTCAG,ACACA AACCCATA~;A:GAGGATTXCA:ACAGTACTCT CCGGGTGGTC 1050 AGTGCCCTCC:CCATCCAGCA:~:CCAGGACTGG ATGAGTGGCA~,AGGAGTTCAA 1100 ~
ATG ~ TC~A~,ACAACAAAG ACCTCCCAGC:GCCCATCGAG~AGAACCATCT 1150 ~`-.,,.':
A,¢GGTCAGTA AGAGCTCCAC:AGGTATATGT CTTGCCTCCA 1200 .
:'CCAGAAG~ G AGATGACTAA GAAACAGGTC'ACTCTGACCT:GCATGGTCAC 1250 ,,}."',``, :AG ~ T~A~TG:~CCT ~ ACA:TTTACGTGGA GTGGACCAAC AACGGGAAAA 1300 .~' CAG M CTAAA~CTACAAGAAC ACTGAACCAG TCCTGGACTC TGATGGTTCT 1350 .'~
TACTTCATGT~ACAGCAAGCT~GAGAGTGGAA~AAGAAGAACT GGGTGGAAAG 1400 ;'~'`
AAATAGCTAC~:TCCTGTTCAG TGGTCCACGA GGGTCTGCAC AATTACCACA 1450 .~
CGACTAAGAG~CTTCTCCCGG ACTCCGGGTA AATGAGCTCA GCACCCACAA 1500 .,:
~ AACTCTCAGG:TCCAAAGAGA CACCCACACT CATCTCCATG CTTCCCTTGT 1550 .:'.'`-~
M~ :ATAAATAAAG::~CACCCAGCAA TGCCTGGGAC CATGTAAAAA AAA~UUUUAAA 1600 .~
AAAAAACGGG:~ATCCTCTAGA GTCGACCTGC AGGCA 1645 '.'.
~ ::
, (2) INFORMATION FOR SEQ ID NO: 4 (i) SEQUENCE CNARACTERISTICS~
(A) LENGTH: 477 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single Stranded : .
:
:: ~ : `:
, ~ ' `'. ~
~: :
..:
, ~.

J ~ ij PCl'/US93/05709 (D) TOPOLOGY: Unknown (ii) MOLECULE TYPE: Nucleic Acid ~:~
(iii) HYPOTHETICAL: Not Applicable (iv) ANTI-SENSE: Not Applicable (v) FRA&MENT TYPE: Not Applicable (vi) ORIGINAL SOURCE: Synthetically Prepared -~
(vii) IMMEDIATE SOURCE: Synthetically Prepared : ~viii) POSITION IN GENOME: None ~-;
: (ix) FEATURE: None : (x) PUBLICATION INFORMATION: None ~i.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

AASGNTNSSY AMSWVRNTNN KRNNWVASNS SDGNTNYVDS VKGRNTVSRD 100 :~
NARNNNYNNM~SSNRSNDTAM YYCARNDYYG:GGGNGYWGNG TNATVSAAKT 150 '~
:~: TANSVY ~ ~VCGDTTGSSV TNGCNVKGYN NNNVTNTWNS~GSNSSGVHTN 200 `.
- ~- NAVNNS~DNYT:NSSSVTVT9S~TWNSNSNTCM VAHNASSTKV~DKKNNNRGNT 250 NgNCNNCKCN~ANN-NNGGNSV~NNNNNKNKDV ~ SNSNNVT C W VDVSNDD 300 DVNy5wNvN NvNvHTAN~N THRNDYN5TN R WSANNNNH NDWMSGKNNK 350 `,:
C ~ ~:NNNRTNSK~K GS:VRANNVYV NNNNNNNMTX KNVTNTCMVT 400 D~ ~ ~ ~ KT~NNN YKNTNNVNDS DGSYNMYSKN RVNRKNWVNR450 NSYSCSVVHN~GNHNY TTKS NSRTNGK 477 ~

(2) INFO~MATION~FOR SEQ ID NO: 5: l i) SEQUENCE~CNARACTERISTICS~
(A) LENGTH: 1041 (B) TYPE: Nu¢leic Acid (C) ~STRANDEDNESS: Single Stranded:
(D) TOPOLOGY: Unknown :~
ij MOLE~ULE TYPE!~ Nucleic Acid (iii) HYPOTHETICAL: Not Applicable : ~ (iv) ANTI-SENSE: Not Applicable -~-:(v):PRAGMENT~TYPE: Not Applicable : : ~
~ .

~

WO93/2237 ~ PCT/US93/05709 :~, .. . .
--19-- .:
(vi) ORIGINAL SOURCE: Synthetically Prepared ;~
(vii) IMMEDIATE SOURCE: Synthetically Prepared . :
(viii) POSITION IN GENOME: None . ~,, (ix) FEATU~E: None (x) PUBLICATION INFORMATION: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5~

GGTCTTTGTA TACATGTTGC TGTGGTTGTC TGGTGTTGAT GGAGACATTG 100 '-`.'.'~:.

AGCGTCACCT GCAAGGCCAG TCAAAATGTG CATACTAATG TTGCCTGGTA 200 ."'.i TCAACAGAAA CCAGGACAAT CTCCTAAAGC ACTGATTTAC TCGGCATCCT 250 '::~'.~:.i.
ACCGTTACAG TGGAGTCCCT GATCGCTTCA CAGGCAGTGG ATCTGGGACA 300 '~'-'``' :GATTTCACTC ~CACCATCAG CAkTGTGCAG TCTGAAGACT TGGCAGAATA350 ,'~' . TTTCTGTCAG CAATGTAACA:GCTATCCTCT~ATTCACGTTC GGCTCGGGGA 400 j~,.!,`~'`CAACGTTGGA AATAAAACGG GCTGATGCTG CACCAACTGT ATCCATCTTC 4S0 ~.
CCACCATCCA GTGAGCAGTT AACATCTGGA GGTGCCTCAG TCGTGTGCTT S00 ~.
CTTGAACAAC TTCTACCCCA AAGACATCAA TGTCAAGTGG AAGATTGATG S50 , :GCAGTGAAC:G ACAAAATGGC GTCCTGAACA GTTGGACTGA TCAGGACAGC 600 : ::AAAGACAGCA~CCTACAGCAT GXGCAGCACC CTCACGTTGA CCAAGGACGA 6SO
GTATGAACGA~CATAACAGCT-ATACCTGTGA GGCCACTCAC:AAGACATCAA 700 CTT~ACCCAT~TGTCAAGAGC~:TTCAACAGGA~ATGAGTGTTA GAGACAAAGG750 '~
~ TCCTGAGACG~CCA,CCACCXG~'CTCCCCAGCT~CCATCCTATC TTCC¢TTCTA800 A~ ~ GT ~ ~GGCTTCCCCA~:CAAGCGACCT~ACCACTGTTG:::CGGTGCTCCA850 is,~
¢C~C ~ CTCCTT;CTCCTCCTCC TCCC ~ CCT:TGGCTTTTAT 900 ~'',-'`;' ATATTCAATA~AAGTGAGTCT ~TGCACTTGA 950 AAAAAAAAAA:AAaAAAAAAA~AAAAAAAAAA~AAAAAAAAAA 1000 AAGGGGATCC~TCTAGAGTCG ACCTGCAGGC A ~ 1041 ',`,~

(2j INFORMATION FOR~SEQ ID;NO: 6:
(i) SEQUENCE~CHARACTERISTICS: ;-',,' (A) ~ LENGTH: 23~5:: , ., (B) TYPE: Nucleic Acid :(C) STRANDEDNESS: Single Stranded (D) TOPO ~Gyl- ~Unknown :
-(ii) MOLECULE TYPE: Nucleic Acid -~.
. ~
: (iii) HYPOTHETICAL: Not Applicable (iv) ANTI-SENSE: Not Applicable .

'' : ' :~ ~

W093/2S237 ,~li PCT/US93/05709 -20~
(v) FRAGMENT TYPE: Not Applicable , (vi) ORIGINAL SOURCE: Synthetically Prepared (vii) IMMEDIATE SOURCE: Synthetically Prepared ~`
(viii) POSITION IN GENOME: None (ix) FEATURE: None ~-(x) PUBLICATION INFORMATION: None :
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
MNSNTNVNVY MNNWNSGVDG DNVNTNSNKN MSTSVGGTVS VTCKASNNVH 50 ;~
TNVAWYNNKN GNSNXANNYS ASYRYSGVND RNTGSGSGTD NTNTNSNVNS 100 ~H;

(2) INFORMATION FOR SEQ ID NQ: 7~
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 957 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single Stranded ) TOPOLOGY: Unknown ~;
: ~(ii) MOLECULE TYPE: Nucleic Acid ::
(iii) HYPOTHETICAL: Not Applicable ~' (iv) ANTI-SENSE: Not Applicable (v) FRAGMENT TYPE: Not Applicable (~i) ORIGINAL SOURCE: Synthetically Prepared ~:
(vii) IMMEDIATE SOURCE: Synthetically Prepared :
(~iii) POSITION IN GENOME~ None i " ~ ' ! I I , i ' ' ! , , - (ix) FEATURE: None ~: (x) PUBLICATION INFORMATION: None ,.-(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

.

W O 93/25237 PC~riUS93/05709 TTTCTGTCAG CAATGTAACA GcTATccTcT ATTCACGTTC GGCTCGGGGA 400 CGCCCGTCAC AAAGAGC~TTC AACAGGGGAG AGTGTTAGAG GGAGAAGTGC 750 CCCCACCTG~ TCCTCAGTTC CAGCCTGACC CccTcccATc CTTTGGCCTC 800 TGACCCTTTT TCCACAGGGG:::ACCTACCCCT ATTGCGGTCC TCCAGCTCAT 850 CTTTCACCTC:ACCCCCCTC~C TCCTCCTTGG CTTTAATTAT GCTAATGTTG 900 . ~, (2) INFORMATION FOR SEQ ID NO: 8:
SEQUENCE CHARACTERISTICS:
(A);~LENGTH~ 234 B)~ :TYPE::~ Nucleic Acid ~ ~ ' (Cj ~STRANDEDNESS: Single Stranded - .
(D) :TOPOL0GY: Unknown (ii) MOLECULE~TYPE: Nucleic Acid :
HY OTHETICAL: Not Applicable (iv)~`ANTI-SENSE~ Not Applicable ~`
(v)~ FRAGMENT~TYPE: Not Applicable -:~:
(vi)~ ORIGINAL SOURCE:; Synthetically Prepared (vii) IMMEDIATE SOURCE: Synthetically Prepared , (vi'ii) POSITION IN GENOME: None i ! i ~ :
(ix):FEATURE:~ Non- j:
x) PUBL~CATION INFORMATION: None :
: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: ~
~ .

:: ~:: `:`:

WO93/25~37 ~ PCT/US93/05709, TNVAWYNNKN GNSNKANNYS ASYRYSGVND RNTGSGSGTD NTNTNSNVNS100 ;:~
NDNANYNCNN CNSYNNNTNG SGTTNNNKTV AANSVNNNNN SDNNNKSGTA150 .-~

;
~2) INFORMATION FOR SEQ ID NO: 9~
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1641 (B~ TYPE: Nucleic Acid (C) STRANDEDNESS: Single Stranded (D) TOPOLO~Y: Unknown ..
(ii) MOLECULE TYPE: ~ucleic Acid (iii) HYPOTHETICAL: Not Applicable ~.
(iv) ANTI-SENSE: Not Applicable (v) FRAGMENT TYPE: Not Applicable .:, `(vi ? ORIGINAL SOURCE: Synthetically Prepared (vii) IMMEDIATE SOURCE: Synthetlcally Prepared (viii~ POSITION IN GENOME: None ~ix) FEATURE: None . ~ (x) PUBLICATION INFORMATION: None (xi) SEQUENCE DESCRIPTION. SEQ ID NO: 9:
TTACGAATTC GAGCTCGGTA~CCCCCTGGAT TTGAGTTCCT CACATTCAGT 50 GATG~GCACT GAACACAGAC ACCTCACCAT GAACTTCGGG TTCAGCCTGA 100 TGCAGCCTCC GGATTCACTT TCAGTAGTTA TGCCATGTCT TGGGTTCGCC 250 :

~ATCACCTTCT ATGTAGACAG TGTGAAGGGC CGATTCACCG TCTCCAGAGA 350 CAATGCCAGG AACATCCTGTiACCTGCAAAT GAGCAGTCTG AGGTCTGAGG '400 GGGGCACAGC GGCCCTGGGC TGCCTGGTCA AGGACTACTT CCCCGAACCG 600 ;

- CCCGGCTGTC CTACAGTCCT CAGGACTCTA CTCCCTCAGC AGCGTGGTGA 700 ~:-~ CCGTGCCCTC CAGCAGCTTG GGCACCCAGA CCT~CATCTG CAACGTGAAT 750 ~,.

`;`:~;

W093~25237 ~ c~ PCT/'US93/05709 i;~

-23- , ,.

GACCGTCA,GT CTTCCTCTTC CCCCCAAAAC CCAAGGACAC CCTCATGATC 900 ,.
TCCCGGACCC CTGAGGTCAC ATGCGTGGTG GTGGACGCGA GCCACGAAGA 950 '"~
CCCTGAGGTC AAGTTCAACT GGTACGTGGA CGGCGTGGAG GTGCATAATG 1000 .~, AGCGTCCTCA CCGTCCTGCA CCAGGACTGG CTGAATGGCA AGGAGTACAA 1100 ,?.

TCCCGGGATG AGCTGACCAA GAACCAGGTC AGCCTGACCT GCCTGGTCAA 1250 ,`r'`,'.'.`, CGGAGAACAA CTACAAGACC ACGCCT CG TGCTGGACTC CGACGGCTCC 1350 .,, ~: TTCTTCCTCT ACAGCAAGCT CACGTGGAC AAGAGCAGGT GGCAGCAGGG 1400 `~
GAACGTCTTC TCATGCTCCG:TGATGCATGA GGCTCTGCAC AACCACTACA 1450 ~,,'~`'~`
CGCAGAA6AG CCTCTCCCTÇ TCTCCGGGTA AATGAGTGCG ACGGCCGGCA 1500 AGCCCCCGCT C'CCCGGGCTC TCGCGGTCGC ACGAGGATGC TTGGCACGTA 1550 .'.-,`.
CCCCCTGTAC ATACTTCCCG'GGCGCC~AGC:ATGGGAATAA AGCACCCAGC 1600 GCTGCCCTGG GCCCCTGCAA GGATCCAAGC TTGGCACTGG C 16~1 ,.':, ;``
(2) INFORMATION:FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS: ~S~' : ~ .
- (A) LENGTN 477 (B~) TYPE~ Nucleic Acid ~C~ STRANDEDNESS: Single Stranded '~
(D:)::TOPOLOGY: Unknown ( ii ) MOLECULE TYPE: Nucleic Acid HYPOTHETICAL: Not'Applicable `'~
:(iv~ ANTI~-SENSE::~Not Applicable v)~FRAGMENT-TYPE:~ Not Applicable (vi) ORIGINAL SOURCE: Synthetically Prepared (vi~i) INMEDIATE~SOUR OE : Synthetically Prepared ~-' (viii) POSITION IN GENONE: None ~ ~ -x) 'FEATURE- None ~
: ~ 1.. .:.:
x) PUBLICATION }NFORMATION: None ,~":~ (xi)~ 5E0UENCE: DESCRIPTION: SEQ ID NO: 10:

" ~"~
~, ,-,, ~ -., ,~ ~ j ..

-: : , :

~ 3/25237 ~L~) PCT/US93/05709 ~ ~
;",,;, ...

MSTNHRHNTM NNGNSNNNNV NVNKGVNCNV KNVNSGGGNV KNGGSNKNSC 50 .
AASGNTNSSY AMSWVRNTNN KRNNWVASNS SDGNTNYVDS VKGRNTVSRD 100 ~
NARNNNYNNM SSNRSNDTAM YYCARNDYYG GGGNGYWGNG TNATVSAAST 150 ~'``
KGNSVNNNAN SSKSTSGGTA ANGCNVKDYN NNNVTVSWNS GANTSGVHTN 200 ~`~
NAVNNSSGNY SNSS W TVNS SSNGTNTYNC NVNHKNSNTK VDKKVNNKSC 250 `~

GNYNSDNAVN WNSNGNNNNN YKTTNNVNDS DGSNNNYSKN TVDKSRWNNG 450 - ~;
NVNSCSVMHN ANHNHYTNKS NSNSNGK 477 ; `' "'~'.;'~'.

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Claims (6)

WE CLAIM:

1. A chimeric murine-human T84.12 antibody the kappa gene and the gamma gene of said antibody each having a murine variable region and a human constant region.

2. A chimeric murine-human T84.12 antibody kappa gene having a murine variable region and a human constant region.

3. A chimeric murine-human T84.12 antibody gamma gene having a murine variable region and a human constant region.

4. Isolated DNA having the sequence depicted by SEQ ID NO. 7 or SEQ ID NO. 9.

5. Isolated DNA having the sequence depicted by SEQ ID NO. 5.

6. SP2/0 myeloma cells cotransformed with expression vectors including SEQ ID NO. 3 and SEQ ID
NO. 5.