Title:
HUMAN POLYCLONAL ANTIBODIES FROM TRANSGENIC NONHUMAN ANIMALS
Document Type and Number:
WIPO Patent Application WO/2000/046251
Kind Code:
A2
Abstract:
Substantially human antisera are provided by genetically modifying a domestic animal generally weighing at least about 1 kg. The domestic animal is genetically modified by generating inactive heavy and light chain immunoglobulin loci and integrating at least funtional portions of the human heavy and light chain immunoglobulin loci, whereby the human loci generate an immune response. The antisera find use in the treatment of diseases, immunocomprised patients and in case of transplantation.
Inventors:
BUELOW JENS-ULRICH (DE)
Application Number:
PCT/EP2000/000933
Publication Date:
August 10, 2000
Filing Date:
February 04, 2000
Assignee:
BUELOW JENS ULRICH (DE)
International Classes:
A61K39/395; A61P31/00; A61P35/00; A61P37/00; C07K16/00; C07K16/06; A01K67/027; C07K16/08; C12N5/10; C12N15/09; C12N15/85; C12N15/877; (IPC1-7): C07K16/08; C07K16/18; C07K16/28; C07K16/30; C07K16/24; A61K39/395
Domestic Patent References:
Other References:
WEIDLE U H ET AL: "Genes encoding a mouse monoclonal antibody are expressed in transgenic mice, rabbits and pigs." GENE, (1991 FEB 15) 98 (2) 185-91. , XP000919055
FIRST N L: "New animal breeding techniques and their application." JOURNAL OF REPRODUCTION AND FERTILITY. SUPPLEMENT, (1990) 41 3-14. REF: 103 , XP000941129
See also references of EP 1151010A2
Attorney, Agent or Firm:
Ottevangers S. U. (Vereenigde Nieuwe Parklaan 97 BN The Hague, NL)
Ir, Prins A. W. C. S. (Vereenigde Nieuwe Parklaan 97 BN The Hague, NL)
Claims:
| 1. |
accepted by the patient. Administration of the effective dosage may occur in a single infusion or in a series of infusions. Repeated infusions may be administered once a day, once a week once a month, or once every three months, depending on the halflife of the antibody preparation and the clinical indication. For applications on epithelial surfaces the antibody preparations are applied to the surface in need of treatment in an amount sufficient to provide the intended end result, and can be repeated as needed. The antibody preparations find use in their ability to bind and neutralize antigenic entities in human body tissues that cause disease or that elicit undesired or abnormal immune responses. An"antigenic entity"is herein defined to encompass any soluble or cellsurface bound molecules including proteins, as well as cells or infectious disease causing organisms or agents that are at least capable of binding to an antibody and preferably also are capable of stimulating an immune response. Administration of an antibody preparation against an infectious agent as monotherapy or in combination with chemotherapy results in elimination of infectious particles. A single administration of antibodies decreases the number of infectious particles generally 10 to 100 fold, more commonly more than 1000fold. Similarly, antibody therapy in patients with malignant disease as monotherapy or in combination with chemotherapy reduces the number of malignant cells generally 10 to 100 fold, or more than 1000fold. Therapy may be repeated over an extended amount of time to assure the complete elimination of infectious particles, malignant cells, etc. In some instances, therapy with antibody preparations will be continued for extended amounts of time in the absence of detectable amounts of infectious particles or undesirable cells. Similarly, the use of antibody therapy for the modulation of immune responses may consist of single or multiple administrations of therapeutic antibodies. Therapy may be continued for extended amounts of time in the absence of any disease symptoms. The subject treatment may be employed in conjunction with chemotherapy at dosages sufficient to inhibit infectious disease or malignancies. In autoimmune disease patients or transplant recipients antibody therapy may be employed in conjunction with immunosuppressive therapy at dosages sufficient to inhibit immune reactions. The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL Generation of transgenic rabbits expressing substantially human immunoqlobulin Exons encoding human constant region elements and variable region elements are integrated into the genome of rabbit fibroblasts by homologous recombination. Rabbit fibroblasts are transfected with various linearized DNA constructs containing human immunoglobulin locus elements. Successfully transfected cells are selected and used for the cloning of rabbits. Cloning of rabbits Mature Dutch Belton rabbits are superovulated by subcutaneous injection of follicle stimulating hormone (FSH) every 12 hours (0.3 mg x 2 and 0.4 mg x 4). Ovulation is induced by intravenous administration of 0.5 mg luteinizing hormone (LH) 12 hours after the last FSH injection. Oocytes are recovered by ovidual flush 17 hours after LH injection. Oocytes are mechanically enucleated 1619 hours after maturation. Chromosome removal is assessed with bisBENZIMIDE (HOECHST 33342, Sigma, St. Louis, MO) dye under ultraviolet light. Enucleated oocytes are fused with actively dividing fibroblasts by using one electrical pulse of 180 V/cm for 15 us (Electrocell Manipulator 200, Genetronics, San Diego, CA). After 35 hours oocytes are chemically activated with calcium ionophore (6 uM) for 4 min (# 407952, Calbiochem, San Diego, CA) and 2 mM 6dimethylaminopurine (DMAP, Sigma) in CR2 medium (Specialty Media, Lavalett, NJ) with 3 mg/ml bovine serum albumin (fatty acid free, Sigma) for 3 hours. Following the activation, the embryos are washed in hamster embryo culture medium (HECM)Hepes five times and subsequently, cultivated in CR2 medium containing 3 mg/mgl fattyacid free BSA for 7 days at 37.8° C and 5% C02 in air. Embryos are then transferred into synchronized recipients. Offsprings are analyzed by PCR for a segment of the transgene. Binding of human antibodies expressed in rabbits to Hepatitis B surface antigen Genetically engineered rabbits (as described above) are immunized intramuscularly with purified Hepatitis B surface antigen (HBsAg) (long in incomplete Freund's adjuvant) on day 0 and day 14. On day 28 animals are bled from the ear and serum is prepared. ELISA plates (NUNC, Denmark) are coated with 1 Hg/ml HBsAg in PBS for 1 hour at room temperature. Subsequently, available binding sites are blocked by incubation with 1% nonfat dry milk (NFM) in PBS (300 pl/well). Rabbit serum is diluted in PBS/1% NFM and added to the coated wells. After an incubation of 1 hour, the plates are washed 3 times with PBS/0.05% Tween 20 and bound Ig is detected using goat antihuman Ig conjugated with horseradish peroxidase. Conjugated goat antibody is detected using ophenylenediamine dihydrochloride (Sigma) at 1 mg/ml. The colorimetric reaction is stopped by addition of 1 M HC1 solution and the absorbance is measured at 490 nm. As a control serum from nonimmunized rabbits is used. Serum from nonimmunized rabbits does not react with HBsAg. At a dilution of 1: 100 the optical density measured in uncoated and HBsAg coated wells is below 0.4. In contrast, serum from immunized rabbits contains substantially human antibodies reactive with HBsAg. At a serum dilution of 1: 100 the measured optical density is 2.8. Upon further dilution of the serum the measured optical density declines to 0.2 (at a dilution of 25600). No antibodies reactive with a goat antirabbit IgGHRP conjugate can be detected. This demonstrates that the genetically engineered rabbits produce substantially human antiHBsAg antibodies following immunization. |
| 2. |
Complement mediated cytotoxicity of virus infection cell line using human antibodies A human liver carcinoma cell line expressing HBsAg is labeled with 0. |
| 3. |
mCi 51Cr in 100 ul PBS for. |
| 4. |
hr at 37°C. Two thousand"Crlableled cells are incubated with serum from genetically engineered rabbits expressing antiHBsAg immunoglobulin (see above). After two hours at 37°C the release of 51Cr into the supernatant is determined by measuring radioactivity using a scintillation counter. For the determination of maximum release, 1% Triton X100 is added. The degree of cell lysis is calculated as follows: % Lysis = CPM experimental CPM#spontaneous/CPM# total CPM spontaneous. Incubation of labeled cells with serum (diluted 1: 30) from nonimmunized rabbits does not result in cell lysis (<10%). However, incubation of cells with serum from immunized rabbits causes 80% cell lysis. Inactivation of complement in the serum by heat treatment (56°C for 30 minutes) renders the serum from immunized rabbits inactive. These results demonstrate that substantially human antibodies produced by genetically engineered rabbits bind to HBsAgpositive cells and cause complement dependent lysis. Treatment of animal with infection. Substantially human immunoglobulin is purified from the serum of genetically engineered rabbits by ammonium sulfate precipitation and ion exchange chromatography. SCIDmice are injected with one million human liver carcinoma cells expressing HBsAg. Subsequently, 25 Hg immunoglobulin is injected peritoneally once per day. Animals treated with antibodies isolated from nonimmunized rabbit serum die after about 60 days. This is similar to untreated recipients of liver carcinoma cells. In contrast, mice treated with antibodies isolated from immunized rabbit serum survive for more than 150 days. This demonstrates that human antibodies produced in genetically engineered rabbits are capable of eliminating human carcinoma cells from SCIDmice. It is evident from the above results that by using genetically engineered rabbits expressing substantially human immunoglobulin genes, polyclonal antibody preparations against antigens, infectious particles, cancer cells, and the like can be generated. Such polyclonal antibody preparations can be used to treat patients suffering from an infectious disease or a malignancy. The antisera also can be used to modulate an immune response by elimination of cell sub populations, cytokines, or the like. The human antibody preparation has a substantially reduced likelihood of engendering an immune response in human patients, as compared to heterologous antisera, it will have few side effects and it can be used safely with positive results. All of the references cited herein are incorporated herein by reference as if each reference was individually wholly incorporated. It will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. CLAIMS 1. A polyclonal antisera composition of a nonhuman animal that specifically recognizes an immunogen, wherein said antisera composition is comprised predominantly of substantially human immunoglobulin protein molecules comprised of at least a portion of a human heavy chain polypeptide, wherein said substantially human immunoglobulin protein molecules specifically bind to said immunogen. |
| 5. |
The polyclonal antisera according to Claim 1, wherein said transgenic nonhuman animal is immunized with said antigenic entity, weighs at least 1 kg and comprises at least a portion of functional human heavy chain immunoglobulin genes integrated by homologous recombination into its genome. |
| 6. |
The polyclonal antisera composition according to Claim 1, wherein said transgenic nonhuman animal generates antibody diversity predominately by gene conversion. |
| 7. |
The polyclonal antisera composition according to Claim 1, wherein said transgenic nonhuman animal is from the order Lagomorpha. |
| 8. |
The polyclonal antisera composition according to Claim 1, wherein said portion of functional human heavy chain immunoglobulin genes comprises at least one constant region element. |
| 9. |
The polyclonal antisera composition according to Claim 5, wherein said portion of functional human heavy chain immunoglobulin genes further comprises at least one variable region element. |
| 10. |
The polyclonal antisera composition according to Claim 6, wherein said variable region element is the variable region element proximal to the D region. |
| 11. |
The polyclonal antisera composition according to Claim 1, wherein said immunogen comprises a disease causing organism or antigenic portion thereof. |
| 12. |
The polyclonal antisera composition according to Claim 1, wherein said immunogen is an antigen endogenous to humans. |
| 13. |
The polyclonal antisera composition according to Claim 1, wherein said immunogen is an antigen exogenous to humans. |
| 14. |
A transgenic nonhuman animal weighing at least 1 kg and comprising at least a portion of functional human heavy chain immunoglobulin genes integrated by homologous recombination into its genome, wherein said portion of functional human heavy chain immunoglobulin genes rearranges in frame with heavy chain immunoglobulin sequences endogenous to said nonhuman animal to encode functional, substantially human antibody molecules that comprise at least in part human heavy chain immunoglobulin polypeptide sequences, and wherein said animal predominantly produces said functional, substantially human antibody molecules when immunized. |
| 15. |
A transgenic nonhuman animal weighing at least 1 kg and comprising at least a portion of functional human light chain immunoglobulin genes integrated by homologous recombination into its genome, wherein said human light chain immunoglobulin genes rearrange in frame with sequences endogenous to said nonhuman animal to encode functional, substantially human antibody molecules that comprise at least in part human light chain immunoglobulin polypeptide sequences. |
| 16. |
The transgenic nonhuman animal according to Claim 11 or 12, wherein said transgenic nonhuman animal generates antibody diversity predominately by gene conversion. |
| 17. |
The transgenic nonhuman animal according to Claim 11 or 12, wherein said transgenic nonhuman animal is from the order Lagomorpha. |
| 18. |
The transgenic nonhuman animal according to Claim 11 or 12, wherein said portion of functional human heavy chain immunoglobulin genes comprises at least one constant region element. |
| 19. |
The transgenic nonhuman animal according to Claim 15, wherein said portion of functional human heavy chain immunoglobulin genes further comprises at least one variable region element. |
| 20. |
The transgenic nonhuman animal according to Claim 16, wherein said variable region element is the variable region element proximal to the D region. |
| 21. |
The transgenic nonhuman animal according to Claim 12, wherein said human immunoglobulin light chain gene encodes the K chain. |
| 22. |
An antisera composition produced by the transgenic nonhuman animal according to Claim 11. |
| 23. |
A method for neutralizing an antigenic entity in a human body component, said method comprising: contacting said body component with an antisera composition according to Claim 1, whereby said substantially human immunoglobulin protein molecules in said antisera composition specifically bind and neutralize said antigenic entity. |
| 24. |
The method according to Claim 20, wherein said antigenic entity is from an organism that causes an infectious disease. |
| 25. |
The method according to Claim 20, wherein said antigenic entity is a cell surface molecule. |
| 26. |
The method according to Claim 22, wherein said cell surface molecule is from a lymphocyte or an adipocyte. |
| 27. |
The method according to Claim 20, wherein said antigenic entity is a human cytokine or a human chemokine. |
| 28. |
The method according to Claim 20, wherein said antigenic entity is a cell surface molecule on a malignant cancer cell. |
| 29. |
A method of producing a transgenic nonhuman animal weighing at least 1 kg and comprising human immunoglobulin genes integrated by homologous recombination into its genome, wherein said animal predominantly produces functional, substantially human antibody molecules comprised at least in part of human immunoglobulin polypeptide sequences when immunized, said method comprising: producing a first mutated animal comprising heavy chain immunoglobulin loci where constant and/or variable region elements are replaced with at least a functional portion of the human heavy chain immunoglobulin locus by genetic alteration of a cell nucleus of said animal, introducing said cell nucleus into an enucleated nuclear transfer unit cell to provide a first embryonic stem cell, introducing said first nuclear transfer unit cell into a female recipient host to produce a first mutated neonate; producing a second mutated animal comprising light chain immunoglobulin loci where constant and/or variable region elements are replaced with at least a functional portion of the human light chain immunoglobulin locus by genetic alteration of a cell nucleus of said animal, introducing said cell nucleus into an enucleated nuclear transfer unit cell to provide a second embryonic cell stem cell, introducing said second nuclear transfer unit cell into a female recipient host to produce a second mutated neonate; and breeding mature first and second mutated neonates and selecting animals capable of producing substantially human antisera and being at least substantially incapable of producing endogenous antisera. |
| 30. |
A method of producing a transgenic nonhuman animal weighing at least 1 kg and comprising human immunoglobulin genes integrated by homologous recombination into its genome, wherein said animal predominantly produces functional, substantially human antibody molecules comprised at least in part of human immunoglobulin polypeptide sequences when immunized, said method comprising: producing a mutated animal comprising heavy and light chain immunoglobulin loci where constant and/or variable region elements are replaced with at least a functional portion or the human heavy and/or light chain immunoglobulin locus by genetic alteration of a cell nucleus of said animal, introducing said cell nucleus into an enucleated nuclear transfer unit cell to provide a embryonic cell stem cell, introducing said nuclear transfer unit cell into a female recipient host to produce mutated neonate; and breeding mature mutated neonates and selecting animals capable of producing substantially human antisera and at least substantially incapable of producing endogenous antisera. |
| 31. |
The method according to Claim 26 or 27, wherein said nuclear transfer unit cell is an oocyte. |
| 32. |
The method according to Claim 26 or 27, wherein said animal is from the order of Lagomorpha. |
| 33. |
A method according to Claim 26 or 27, wherein said heavy chain locus comprises at least one constant region element. |
| 34. |
A method according to Claim 26 or 27, wherein said heavy chain locus comprises at least one variable region element. |
| 35. |
A method according to Claim 26 or 27, wherein said heavy chain locus comprises the variable region element proximal to the D region. |
Description:
INTERNATIONALSEARCHREPORT In : itionalApplicationNo PCT/EP00/00933 C.(Continuation)DOCUMENTSCONSIDEREDTOBERELEVANT Category°Citationofdocument,withindication,whereappropriate
,oftherelevantpassagesRelevanttoclaimNo. AFIRSTNL:"Newanimalbreeding1-32 techniquesandtheir application." JOURNALOFREPRODUCTIONANDFERTILITY. SUPPLEMENT,(1990)413-14.REF:103, XP000941129 thewhole document 2 .,ternationalapplicationNo. INTERNATIONALSEARCHREPORTPCT/EP00/00933 Box)Observationswherecertainclaimswerefoundunsearchable(Cont
inuationofitem1offirstsheet) ThisInternationalSearchReporthasnotbeenestablishedinrespecto
fcertainclaimsunderArticle17(2)(a)forthefollowingreasons: 1.FXIClaimsNos.: becausetheyrelatetosubjectmatternotrequiredtobesearchedbythi
sAuthority,namely: Althoughclaims20-25aredirectedtoamethodoftreatmentofthe human/animalbody,thesearchhasbeencarriedoutandbasedonthealle
ged effectsoftheantiseracomposition. 2.mClaims Nos.:1,5-10and20-25(all partly) becausetheyrelatetopartsoftheInternational Applicationthatdonotcomplywiththeprescribedrequirementstosuc
h anextentthatnomeaningfulInternationalSearchcanbecarriedout,s
pecifically: seeFURTHERINFORMATIONsheetPCT/ISA/210 3.ClaimsNos.: becausetheyaredependentclaimsandarenotdraftedinaccordancewit
hthesecondandthirdsentencesofRule6.4(a). BoxIIObservationswhereunityofinventionislacking(Continuation
ofitem2offirstsheet) ThisinternationalSearchingAuthorityfoundmultipleinventionsin
thisinternationalapplication,asfoNows: 1. n As all required additional searchfeesweretimelypaidbytheapplicant,thisInternationalSear
chReportcoversall searchableclaims. 2.F As all searchable claims couldbesearchedwithouteffortjustifyinganadditionalfee,thisAu
thoritydidnotinvitepayment ofanyadditionalfee. 3.As ontysomeoftherequiredadditionalsearchfeesweretimelypaidbythe
applicant,thisInternationalSearchReport coversonlythoseclaimsforwhichfeeswerepaid,specificallyclaims
Nos.: 4.Norequiredadditionalsearchfeesweretimelypaidbytheapplicant
.Consequently,thisInternationalSearchReportis restrictedtotheinventionfirstmentionedintheclaims;itiscovere
dbyclaimsNos.: RemarkonProtestTheadditionalsearchfeeswereaccompaniedbytheap
plicant'sprotest. u n Noprotestaccompaniedthepaymentofadditionalsearchfees. u FURTHERINFORMATIONCONTINUEDFROMPCT/ISA/210 ContinuationofBox1.2 ClaimsNos.:1,5-10and20-25(allpartly) Inclaim1theterm"transgenic"isnotpresentanditisreferredtoa "nonhumananimal"whereasinclaims2-4whicharedependentonsaidcla
im thephrase"Thepolyclonalantiseraaccordingtoclaim1,whereinsaid
transgenicnonhumananimal"hasbeenused,whichmeansthatthenonhum
an animalreferredtoinclaim1shouldbea"transgenic"one. Inviewofthisdiscrepancybetweenclaim1andclaims2-4,andtaking intoconsiderationthefactthatthedescriptionreferstotransgenic
animalsonly,thesearchhasbeenlimitedwithrespecttoclaims1,5-10
and20-25tothoseembodimentswhicharedefinedwithreferenceto transgenicnonhumananimas. Theapplicant'sattentionisdrawntothefactthatclaims,orpartsof claims,relatingtoinventionsinrespectofwhichnointernational searchreporthasbeenestablishedneednotbethesubjectofan internationalpreliminaryexamination(Rule66.1(e)PCT).Theappli
cant isadvisedthattheEPOpolicywhenactingasanInternational PreliminaryExaminingAuthorityisnormallynottocarryouta preliminaryexaminationonmatterwhichhasnotbeensearched.Thisis
thecaseirrespectiveofwhetherornottheclaimsareamendedfollowin
g receiptofthesearchreportorduringanyChapterIIprocedure. INTERNATIONALSEARCHREPORTlr. ational Application No Information on patent family membersPCT/EP00/00933 PatentdocumentPublication PatentfamilyPublication citedinsearchreportdate member(s)date US5814318A29-09-1998US 5661016 A 26-08-1997 US 5545806 A 13-08-1996 US 5789650 A 04-08-1998 US 5569825 A 29-10-1996 AU 6819494 A 21-11-1994 CA 2161351 A 10-11-1994 EP 0754225 A 22-01-1997 JP 8509612 T 15-10-1996 WO 9425585 A 10-11-1994 US 5625126 A 29-04-1997 US 5770429 A 23-06-1998 AU 1318299 A 01-04-1999 AU 3907895 A 29-02-1996 AU 3328493 A 19-07-1993 AU 720612 B 08-06-2000 AU 4190297 A 15-01-1998 CA 2124967 A 24-06-1993 EP 0746609 A 11-12-1996 JP 7503132 T 06-04-1995 WO 9312227 A 24-06-1993 US 5877397 A 02-03-1999 AT 158021 T 15-09-1997 AU 8507191 A 30-03-1992 CA 2089661 A 01-03-1992 DE 69127627 D 16-10-1997 DE 69127627 T 19-02-1998 DK 546073 T 02-02-1998 EP 0546073 A 16-06-1993 EP 0814159 A 29-12-1997 ES 2108048 T 16-12-1997 GB 2272440 A 18-05-1994 GR 3024701 T 31-12-1997 JP 2938569 B 23-08-1999 JP 6500233 T 13-01-1994 KR 241638 B 01-02-2000 WO 9203918 A 19-03-1992 US 5874299 A 23-02-1999 US 5633425 A 27-05-1997 WO9004036A19-04-1990AT 138104 T 15-06-1996 AU 4417389 A 01-05-1990 DE 68926508 D 20-06-1996 DE 68926508 T 31-10-1996 EP 0438474 A 31-07-1991 JP 4500911 T 20-02-1992 KR 164608 B 15-01-1999 US 5545807 A 13-08-1996
