![(12) United States Patent (10) Patent N0.: US 8,283,294 B2 Kastrup Et A]](http://data.docslib.org/img/01c5cf54de42d7d74ec52b9801845a23-1.webp)
US008283294B2
(12) United States Patent (10) Patent N0.: US 8,283,294 B2 Kastrup et a]. (45) Date of Patent: Oct. 9, 2012
(54) METHOD FOR CLONING COGNATE FOREIGN PATENT DOCUMENTS ANTIBODIES FR 2724393 3/1996 W0 WO 92/15678 Al 9/1992 (75) Inventors: Jesper Kastrup, Stenlose (DK); Lars S. W0 WO 93/03151 Al 2/1993 Nielsen, Niva (DK); Per-Johan Meijer, W0 WO 93/20227 A1 10/1993 Copenhagen (DK) W0 WO 94/08008 Al 4/1994 W0 WO 95/20401 Al 8/1995 (73) Assignee: Symphogen A/S, Lyngby (DK) W0 WO 96/08564 Al 4/1996 W0 WO 98/57994 A2 12/1998 ( * ) Notice: Subject to any disclaimer, the term of this W0 WO 99/16904 Al 4/1999 patent is extended or adjusted under 35 W0 WO 99/29888 Al 5/1999 U.S.C. 154(b) by 122 days. W0 WO 01/89563 A1 11/2001 W0 WO 01/92291 A2 12/2001 (21) Appl.No.: 12/074,066 WO 2004/003019 * 6/2004 (22) Filed: Feb. 29, 2008 W0 WO 2004/061104 A2 7/2004 (65) Prior Publication Data W0 WO 2005/042774 A2 5/2005 W0 WO 2006/007850 Al l/2006 US 2008/0227660 A1 Sep. 18, 2008 W0 WO 2006/007853 A2 l/2006 W0 WO 2007/003041 Al l/2007 W0 WO 2007/101441 Al l/2007 Related US. Application Data W0 WO 2007/065433 A2 6/2007 (60) Provisional application No. 60/904,772, ?led on Mar. OTHER PUBLICATIONS 5, 2007. Rudikoff et al (Proc Natl Acad Sci USA 1982 vol. 79 p. 1979).* Ward et al. (Nature 341:544-546 (1989)).* (30) Foreign Application Priority Data Smith-Gill et al. (J. Immunol. 139:4135-4144 (1987)).* Kumar et al. (J. Biol. Chem. 275:35129-35136 (2000)).* Mar. 1, 2007 (DK) ...... 2007 00316 Song et al. (Biochem Biophys Res Comm 268:390-394 (2000)).* Soderlind et al. (Gene 160:269-272 (1995).* (51) Int. Cl. Soderlind et al. (Immunotechnology 4:279-285 (Mar. 1999).* Ardavin, C., et al., “B Cell Response After MMTV Infection: C40B 40/08 (2006.01) Extrafollicular Plasmablasts Represent the Main Infected Population C40B 50/06 (2006.01) and Can Transmit Viral Infection,” J'. Immunol. 162:2538-2545, The (52) US. Cl...... 506/17; 506/26; 424/133.1 American Association of Immunologists (1999). (58) Field of Classi?cation Search ...... 506/17, Babcook, 18., et al., “A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of 506/26; 424/133.1 de?ned speci?cities,” Proc. Natl. Acad. Sci. USA 93:7843-7848, See application ?le for complete search history. National Academy of Science (1996). Bregenholt, S., et al., “Recombinant Human Polyclonal Antibodies: (56) References Cited A New Class of Therapeutic Antibodies Against Viral Infections,” Curr". Pharm. Des. 12:2007-2015, Bentham Science Publishers (Jan. U.S. PATENT DOCUMENTS 2006). Haurum, J.S., “Recombinant polyclonal antibodies: the next genera 4,683,202 A 7/1987 Mullis 5,698,435 A 12/1997 Robinson et al. tion of antibody therapeutics?,” Drug Discov. Today 11:655-660, 5,882,856 A 3/1999 Shuber Elsevier Science (Jul. 2006). 5,912,172 A 6/1999 Eshhar et a1. Liu, A.Y., et al., “Expression of mousezzhuman immunoglobulin 6,096,878 A 8/2000 Honjo et a1. heavy-chain cDNA in lymphoid cells,” Gene 54:33-40, Elsevier Sci 6,111,166 A 8/2000 van de Winkel ence (1987). 6,258,529 B1 7/2001 BerdoZ et al. Liu, M.-L., et al., “Chronic idiopathic myelo?brosis terminating in 6,538,124 B1 3/2003 Idusogie et al. extramedullary anaplastic plasmacytoma,”Leuk. Lymphoma 47:315 6,849,259 B2 2/2005 Haurum et al. 322, Taylor & Francis (Feb. 2006). 6,867,021 B2 3/2005 Maes et a1. 6,994,963 B1 2/2006 Murphy et a1. (Continued) 7,749,697 B2 7/ 2010 OleksieWicZ et al. Primary Examiner * Lynn Bristol 2001/0027249 A1 10/2001 Le et al. 2002/0102604 A1 8/ 2002 Milne Edwards et a1. (74) Attorney, Agent, or Firm * Sterne, Kessler, 2003/0096343 A1 5/ 2003 Robinson et al. Goldstein & Fox P.L.L.C. 2003/0104477 A1 6/ 2003 Buechler et al. (57) ABSTRACT 2003/0175837 A1 9/2003 Le et al. 2004/0067532 A1 4/2004 Zhu et al. The invention relates to a procedure for linking cognate pairs 2005/0180967 A1 8/ 2005 Haurum et al. ofVH and VL encoding sequences from a population of cells 2006/0228350 A1 10/2006 Wu et al. enriched in particular surface antigen markers. The linking 2006/0275766 A1 12/2006 Haurum et al. procedure involves a multiplex molecular ampli?cation pro 2007/0003515 A1 l/2007 Leonard et a1. cedure capable of linking nucleotide sequences of interest in 2007/0141048 A1 6/2007 OleksieWicZ et al. 2008/0069822 A1 3/2008 Jensen et al. connection With the ampli?cation, in particular polymerase 2008/0131882 A1 6/2008 Rasmussen et al. chain reaction (multiplex PCR). The method is particularly 2008/0226630 A1 9/2008 Lantto et a1. advantageous for the generation of cognate pair libraries as 2009/0004192 A1 l/ 2009 Pedersen et al. Well as combinatorial libraries of variable region encoding 2009/0017017 A1 l/2009 Rasmussen et al. sequences from immunoglobulins. The invention also relates 2009/0111142 A1 4/ 2009 Nielsen et al. to methods for generation of chimeric human/non-human 2009/0136498 A1 5/ 2009 Haurum et al. antibodies and expression libraries generated by such meth 2009/0137003 A1 5/ 2009 Tolstrup et al. ods. 2010/0069262 A1 3/ 2010 Nielsen et al. 2010/0310558 A1 12/ 2010 OleksieWicZ et al. 36 Claims, 8 Drawing Sheets US 8,283,294 B2 Page 2
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1 specific for wtEGFR 2 wtEGFR + EGFRvIII 3 speci?c for EGFRvIii {CHEM-phi. 4,non-rsactiva In ELISA US 8,283,294 B2 1 2 METHOD FOR CLONING COGNATE a) amplifying, in a multiplex molecular ampli?cation pro ANTIBODIES cedure, nucleotide sequences of interest using a template derived from a population of genetically diverse cells; This applications claims the bene?t of the ?ling date of b) Wherein the genetically diverse cells are derived from a US. Provisional Appl. No. 60/904,772, ?led Mar. 5, 2007, lymphocyte-comprising cell fraction, from a donor; and DanishAppl. No. PA 2007 00316, ?led Mar. 1, 2007, both c) Wherein at least a subpopulation of the cells express of Which are incorporated by reference in their entirety. CD43 and CD138 antigen or MHCII and B220 antigen; and d) effecting linkage of the nucleotide sequences of interest BACKGROUND OF THE INVENTION ampli?ed in step a). This method provides a combinatorial library of randomly 1. Field of the Invention combined heavy and light chain variable domains. The present invention relates to a procedure for linking Experimental evidence provided in the present application establish that cell populations isolated from mouse spleno cognate pairs of VH and VL encoding sequences from a popu cytes and being positive for the listed surface antigens provide lation of cells enriched in particular surface antigen markers. a good starting material for cloning of antibody encoding The linking procedure involves a multiplex molecular ampli sequences using a multiplex molecular ampli?cation method. ?cation procedure capable of linking nucleotide sequences of The methods of the invention can easily be applied to other interest in connection With the ampli?cation, in particular species expressing the orthologs of CD43 and CD138 or polymerase chain reaction (multiplex PCR). The method is MHCII and B220, in particular the methods can be applied to particularly advantageous for the generation of cognate pair 20 other rodent such as rat. libraries as Well as combinatorial libraries of variable region The method provides several advantages over the altema encoding sequences from immunoglobulin. The invention tive, viZ generation of hybridoma. When preparing hybri also relates to methods for generation of chimeric human/ doma from an immunised mouse, the established cell lines non-human antibodies and expression libraries generated by Will encode a repertoire of different antibody isotypes. The such methods. 25 hybridoma cell lines Will subsequently have to be screened 2. Background Art forboth function (eg binding to a speci?c antigen, ef?cacy in WO 2005/042774 (Symphogen) discloses a method for neutralising a pathogen) and for the antibody isotype. Thus it linking nucleotide sequences of interest in particular cognate requires a tWo step screening procedure to select a hybridoma pairs of VH and VL encoding sequences using a multiplex With a particular antibody isotype and With a particular effect molecularprocedure. The sequences of interest are preferably 30 in a functional assay. Finally, in order to generate a producer ampli?ed and linked from isolated single cells folloWing lim cell line, the antibody secreted by the selected hybridoma Will ited dilution or other cell separation techniques. The refer need to be cloned and sequenced before it can be transferred ence discloses various Ways of enriching a lymphocyte con to a producer cell line. taining cell population to obtain a population of plasma cells Using the method of the present invention, the antibody isotype is determined by the primers used for the multiplex that are particularly suitable for the multiplex molecular molecular ampli?cation, and therefore this Will not need to be ampli?cation procedure. determined. The antibody isotype may be also be determined (and can be changed) by the subsequent linking or splicing of BRIEF SUMMARY OF THE INVENTION constant domain(s) to cloned variable sequences. Further 40 more, the method of the invention provides a library of poly The present invention focuses on methods for generating nucleotides that can be easily sequenced and/or inserted into libraries of immunoglobulin encoding sequences from non vectors, such as expression, transfer, display or shuttle vec human animals and methods for generating in a feW steps tors, so that once a particular antibody has been selected, it is adapted for high throughput cloning and screening, libraries already cloned, its sequence is already knoWn and it can be of vectors coding for chimeric antibodies comprising human 45 transferred easily to an appropriate expression vector for pro constant regions and non-human variable regions. duction of antibody. In a ?rst aspect the invention relates to a method of pro It is expected that the cells sorted according to the provided ducing a library of cognate pairs comprising linked variable protocol provide a source of high a?inity antibodies, poten region encoding sequences, said method comprising: tially With af?nities in the picomolar range. Monoclonal anti a) providing a lymphocyte-comprising cell fraction from a 50 bodies from hybridoma do not possess af?nities in the pico donor; molar range, and Will need to be synthetically a?inity b) obtaining a population of isolated single cells, compris matured to reach these af?nities. ing distributing cells from said cell fraction individually into According to one embodiment these methods further com a plurality of vessels, Wherein at least a subpopulation of the prise assessing prior to multiplex molecular ampli?cation cells express CD43 and CD138 antigen or MHCII and B220 55 that the population of lymphocyte-comprising cells com antigen; and c) amplifying and effecting linkage of the vari prises cells express CD43 and CD138 antigen or MHCII and able region encoding sequences contained in said population B220 antigen, preferably CD43 and CD138. Also the meth of isolated single cells by amplifying, in a multiplex molecu ods may include enriching said lymphocyte-comprising cell lar ampli?cation procedure, nucleotide sequences of interest fraction for a lymphocyte population expressing CD43 and using a template derived from an isolated single cell or a 60 CD138 antigen or MHCII and B220 prior to multiplex population of isogenic cells; and effecting linkage of the molecular ampli?cation. nucleotide sequences of interest ampli?ed. Preferably the methods further comprise isolating from This method provides a library of cognate pair antibodies said lymphocyte-comprising population cells expressing or antibody fragments. CD43 and CD138 antigen or MHCII and B220 antigen prior In another aspect the invention relates to a method of link 65 to multiplex molecular ampli?cation. In a preferred embodi ing a plurality non-contiguous nucleotide sequences of inter ment the isolated cells or subpopulation of cells are CD138 est randomly said method comprising: High/CD43 High or CD 1 38 Intermediate/CD43 High relative US 8,283,294 B2 3 4 to the lymphocyte-comprising cell fraction. More preferably ing of non-human immunoglobulin variable region encoding the isolated cells or subpopulation of cells are CD138 High/ sequences, and human immuno globulin heavy and light chain CD43 High relative to the lymphocyte-comprising cell frac constant regions. tion. Preferably, the vectors are expression vectors enabling the Enrichment or isolation preferably comprises an auto expression of the antibody members of the library for subse mated sorting procedure, such as MACS or FACS. quent screening. More preferably the expression vector is for In a further aspect the invention relates to a method for mammalian expression. generating a vector encoding a chimeric antibody With human The vectors of the library may be obtained by a method of constant regions and non-human variable regions, said the invention. method comprising: In one embodiment the light chain constant region is a a) providing a lymphocyte-comprising cell fraction from a kappa constant region. non-human animal; The non-human sequences may be from rat, sheep, goat, rabbit, guinea-pig or other suitable animal for Which immu b) obtaining a population of isolated single cells, compris nisation protocols have been described, for Which sequence ing distributing cells from said cell fraction individually into information is available to alloW the design of suitable prim a plurality of vessels; ers, and for Which suitable cell sorting techniques enable c) amplifying and effecting linkage of the variable region sorting of plasma cells for single cell mulitiplex molecular encoding nucleic acids contained in said population of iso ampli?cation to link cognate pairs of variable region lated single cells by amplifying, in a multiplex molecular sequences. In one embodiment, the non-human sequences are ampli?cation procedure, said nucleic acids using a template 20 of non-human primate origin, such as cynomolgus monkey, derived from an isolated single cell or a population of Rhesus monkey, chimpanzee, or macaque. Preferably the isogenic cells; and effecting linkage of the ampli?ed nucleic non-human sequences are rodent, such as murine or rat. In acids encoding variable regions of heavy and light chains; another embodiment the non-human sequences are rabbit d) effecting linkage of the ampli?ed variable regions to sequences. human constant regions; and 25 Preferably the variable regions of the antibodies are cog e) inserting the obtained nucleic acid into a vector. nate pairs. Preferably the non-human animal is a mouse. To the extent In another aspect the invention relates to a sub-library that the methods of the invention are applied to mouse cells, Which codes for antibodies exhibiting desired binding speci the methods are named: Mouse-SymplexTM or mSymplexTM. ?cities directed against a particular target, selected from a By this aspect of the invention there is provided a novel 30 library according to the invention. method for generation of libraries of chimeric human/non human antibodies. This is made possible by combining the BRIEF DESCRIPTION OF THE multiplex molecular ampli?cation and subsequent cloning DRAWINGS/FIGURES into a vector backbone With ligation and/ or splicing of human heavy and light chain constant domains. Traditionally, in a 35 FIG. 1. MurineimSymplexTM PCR: Multiplex overlap method for generating chimeric human/non-human antibod extension RT-PCR for the ampli?cation and cognate linkage ies, the chimerisation has been the last step after hybridoma of heavy and light chain antibody genes from a single cell. have been established and screened and the encoded antibody Exemplary primer mixes used for the RT-PCR and the nested has been cloned. Chimerisation may affect the binding speci PCR are described in detail in Table 2 and Table 3 (or Table 5). ?city and/or a?inity of an antibody, and thus there is a risk that 40 FIG. 2. Murine repertoire cloning: a pool of mSymplexTM a good monoclonal mouse antibody loses its ef?cacy When it PCR products encoding VH/VL gene pairs from single is chimerised into a human/mouse antibody. plasma cells Were spliced to the gene encoding human kappa By the provision of a method that directly generates an constant light chain by splicing by overlap extension. The antibody of chimeric antibodies, the screening can be carried pool of genes, encoding complete human-mouse chimeric out on products that do not need to be modi?ed further prior 45 antibodies, Was inserted in an expression vector folloWed by to preclinical and clinical development. an insertion of a bi-directional promoter cassette (2>