United States Patent r191
`Tsuchiya et al.
`
`11•~11111111111111
`5,795,965
`Aug. 18, 1998
`
`US005795965A
`[llJ Patent Number:
`[45J Date of Patent:
`
`[54) RESHAP ED HUMA N TO HUMAN
`INTERLEUK IN-6 RECEPTOR
`
`9109967
`
`711991 WIPO .
`
`OTHER PUBLICATIONS
`
`(75)
`
`Inventors: Masayuki Tuuchiya: Koh Sato. both of
`Gotenba. Japan: Mary Ma.rgaret
`Bendig. West Hamsteaad: Steven
`Tarran J on es. RadJen: J ose William
`Saldanha. Eodfield. all of Great Britain
`
`Oi et al. Biotechniques. vol. 4. No. 3. 1986 p. 214.
`M orrison Hospital Practice Oct. 15. 1989. 65.
`Kitani et al. Clin. Exp. lmmunol. 88. 75-83 1992.
`Suzuki et al. Immunology leners 30( 1991) 17- 22.
`
`(73] Assignee: C hugai Seiyaku Kabushiki Kaisha.
`Tokyo. Japan
`
`[21) Appl. No.:
`(22) PCT Filed:
`
`137,117
`
`Apr. 24, 1992
`
`(86) PCT No.:
`
`PCT/JP92/00S44
`Dec. 20, 1993
`§ 371 Date:
`§ 102(e) Date: Dec. 20, 1993
`
`[87] PCT Pub. No.: W 092/19759
`
`PCT Pub. Date: Nov. 12, 1992
`
`[30)
`
`Foreign Applicatioo Priority Data
`Japan .................................... 3-095476
`Apr. 25. 1991
`[JP]
`Japan .................................... 4-032084
`[JP]
`Feb. 19. 1992
`Int. Cl.6 ..................................................... C07K 16/to
`[51)
`[52) U.S. Cl ................................... 530/387.3; 5301388.24;
`53&388.73
`(58) Field of Seatth ............................ 5301387 3 . 388.22.
`530/388.73
`
`(56)
`
`References Cited
`FOREIGN PATENT DOCUMENTS
`711990 WIPO .
`
`9007861
`
`Primory t.Xominer-Llla Feisee
`Assistam Exmniner-Geetha P. Bansal
`Arrome>; Agent, or Firm-Foley & Lardner
`
`[57]
`
`A BSTRACT
`
`A reshaped human antibody to the human Il..-6R. compris·
`ing:
`(A) an L chain comprising.
`( 1) a human L chain C region. and
`(2) an L chain V region comprising human L chain
`framework regions (FRs). and mouse L chain
`complementary determination regions (CDRS) of a
`momoclonal antibody to the IL-6 recepcor (1L-6R);
`and
`(B) an H chain comprising.
`( 1) a human H chain C region. and
`(2) an H chain V region comprising human H chain
`fRs. and moose H chain CDRs of a monoclonal
`antibody to the Il..-6R.
`
`Since major portion of the reshaped human antibody is
`derived from a human antibody and the mouse C DRs which
`are Jess immunogenic. the present reshaped human antibody
`is less immunogenic to human. a.od therefor is promised for
`therapeutic uses.
`
`(i Claims, 24 Dnwing Sheets
`
`1 of 95
`
`BI Exhibit 1124
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`

`

`U.S. Patent
`US. Patent
`
`Aug. 18, 1998
`Aug. 18, 1998
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`U.S. Patent
`
`Aug. 18, 1998
`
`Sheet 7 of 24
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`5,795,965
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`

`U.S. Patent
`
`Aug. 18, 1998
`
`Sheet 8 of 24
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`5,795,965
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`U.S. Patent
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`Aug. 18, 1998
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`Sheet 9 of 24
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`S,795,965
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`U.S. Patent
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`Aug. 18, 19CJ8
`
`Sheet I 0 of 24
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`U.S. Patent
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`Aug. 18, 1998
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`Sheet 11 of 24
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`Aug. 18, 1998
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`Aug. 18, 1998
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`U.S. Patent
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`Aug. 18, 1998
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`Sheet 16 of 24
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`5,795,965
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`t
`
`RESHAPED ONA FRAGMENT
`
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`U.S. Patent
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`Aug. 18, 1998
`
`Sheet 17 of 24
`
`5,795,965
`
`Fig .17
`
`o-- o
`A ---A
`
`0 ---
`•
`.1.- --
`
`/
`
`/
`
`~/
`•.&7
`fo
`/;/
`~/
`•
`
`1.0
`
`\I)
`0
`'3
`0
`0
`
`0.5
`
`/
`•
`
`. /
`
`- • - STANDARD AUK12-20
`(CHIMERIC)
`- o -
`CHIMERIC AUK12-20
`--A-- RVLa CH I ME RIC H
`
`o~...__._._ _ _._ _ __.__.___._..___.___.__~_.__._
`500
`100
`50
`10
`5
`ANTIBODY CONCENTRATION (ng/mi)
`
`18 of 95
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`

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`U.S. Patent
`
`Aug. 18, 1998
`
`Sheet 18 of 24
`
`5,795,965
`
`Fig .18
`
`-
`
`0
`
`-
`
`STANDARD
`AUK12-20
`
`0.8
`
`0.6
`
`U')
`0
`
`"' 0
`0 0.4
`
`0 .2
`
`0 I 0
`
`O.OL....J..~~..1_____...l___J___.!_L_J._J...J.-1-~~L---l--l-...L-.1..-L-l...-
`10
`50
`100
`500
`ANTIBODY CONCENTRATION (ng/m~)
`
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`U.S. Patent
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`Aug. 18, 1998
`
`Sheet 19 of 24
`
`5,795,965
`
`Fig.19
`
`0.8
`
`0.6
`
`ll'l
`0
`..j
`0
`0
`
`0.4
`
`0.2
`
`- - 0 - - ST AND ARO
`AUK12-20
`---o •--- CHIMERIC
`AUK12-20
`---~ .A ---RVLa+RVHd
`
`0'-----'------'-- --'--'-.L..-..i..-'-'-.l....L------L---'--'-.l-J.-
`10
`50
`100
`500
`ANTIBODY CONCENTRATION (ng/mt)
`
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`U.S. Patent
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`Aug. 18, 19'J8
`
`Sheet 20 of 24
`
`5,795,965
`
`Fig .20
`
`4
`
`3 >
`
`1 >
`< 2
`4
`6
`(SYNTHETIC OLIGONUCLEOT IDr=:s)
`~
`~
`~
`ANNEALING AND EXTENTION AT LAW
`T EMPERATURE USING TAQ ENZYME(FIRST PCR)
`II
`II
`
`A =
`
`~ B
`
`ASSEMBLY AND AMPLTFIING SYNTHETIC GE NE
`AFTER ADDING TERMINAL PRIMERS A ANO B
`(SECOND PCR)
`
`SYNTHETIC GENE
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`21 of 95
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`U.S. Patent
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`Aug. 18, 1998
`
`Sheet 21 of 24
`
`5,795,965
`
`Fig.21
`
`100
`
`50
`
`~---~
`
`/ ---
`y
`jf
`/~
`~ CHI MERIC
`d
`- • - AUK12-20
`~,? --0--MOUSE AUK12-20
`~--~~ -
`A -RVL 0 +RVHs1e122Ha
`
`o,
`
`1000
`100
`10
`ANTIBODY CONCENTRATl ON (ng/m~)
`
`1--i
`
`-0
`
`,.-...,
`0
`.........
`z
`0
`r-
`,_.
`OJ
`.._,
`I
`z ,._.
`
`22 of 95
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`U.S. Patent
`
`Aug. 18, 1998
`
`Sheet 22 of 24
`
`5,795,965
`
`F i g. 22
`
`.........
`
`0 -0 -z
`
`0
`t-...._,
`ro
`.........
`I z
`
`f - -
`
`..........
`
`100
`
`50
`
`0
`
`,
`
`i/
`/io
`CH fMERIC
`.f;/ - • -AUK 12-20
`- -0--MOUSE AUK12-20
`-A - RVL0+RVHs.te1220Hb
`
`i ---~.:::.:=Jlt
`,,.. -
`
`;;;;---
`
`,,,% -
`,,. ~ -;...-
`~~
`
`10000
`
`1tf
`
`/ '
`
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`•
`~a-
`• ". /7
`·~~~
`1000
`100
`10
`ANT I BODY CON CE NT RAT ION ( ng/ ml)
`
`23 of 95
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`U.S. Patent
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`Aug. 18, 1998
`
`Sheet 23 of 24
`
`5,795,965
`
`Fig .23
`
`100
`
`50
`
`- • - CHIMERIC
`AUK12-20
`,..Oa;.;_ie - - 0- - MOUSE AUK 12-20
`,,,""' .,_r
`-A - RVL 0 +RVHsl e 1220Hc
`~~_:~~
`o.__~__._.'--'-'-............... ~_.____.'-'-' ............... .....____.__._~L..L.U.J.__~
`1000
`100
`10
`1
`ANT I BODY CONCENTRATION ( ng/ ml)
`
`,.......
`
`0 -0 ..........
`
`z
`0 ..__.
`f-
`co
`I z .......
`
`24 of 95
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`U.S. Patent
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`Aug. 18, 19CJ8
`
`Sheet 24 of 24
`
`5,795,965
`
`Fig .24
`
`100
`
`50
`
`~ --= ---
`i-~=~·
`~~ --
`-~ -
`j'?'
`-1
`;~-•_ CHIMERfC
`f --o --MOUSE AUK 12-20
`AUK 12-20
`%1f -A - RVt0 +RVHs1e1220Hd
`
`/~/
`1:::::::: o- ..-
`,.0
`"
`.,. .,.
`1000
`100
`10
`ANT I BODY CONCENTRATION (ng/mi)
`
`1
`1
`
`1CXX)O
`
`..........
`
`0
`
`0 -
`' - " z
`0
`t-
`
`........ co
`,__.
`I z
`
`25 of 95
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`5.795.965
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`1
`RESHAPED HUMAN TO HUMAN
`INTERLEUKIN-6 RECEPTOR
`
`TECHNICAL FIELD
`The present invention relates to variable regions (V
`region) of a mouse monoclonal antibody to the human
`interleukin-6 receptor (Il..-6R). human/mouse chimeric anti(cid:173)
`body to the human Il..-6R. and reshaped human antibody
`comprising a human antibody wherein the complementarity
`determining regions (CDRs) of the human light chain (L
`chain) V region and of the human heavy chain (H chain) V
`region are grafted with the CDRs of a mouse monoclonal
`antibody to the human IL-6R. Moreover. the present inven(cid:173)
`tion provides DNA coding for the above-mentioned anti(cid:173)
`bodies or part thereof. The present invention further pro(cid:173)
`vides vectors. especially expression vectors comprising said
`DNA. and host cells transformed or transfected with said
`vector. The present invention still more provides a process
`for production of a chimeric antibody to the human IL-6R.
`and process for production of a reshaped human antibody to
`the human Il..-6R
`
`JO
`
`2
`to bind antigen with the same specificity as the original
`mouse antibody. In addition. chimeric antibodies have a
`substantial reduction in the percent of the p rotein sequence
`derived from a non-human source andl. therefore. are
`expected to be less immunogenic than the original mouse
`antibody. Although chimeric antibodies are predicted to bind
`antigen well and to be less immunogenic. an immune
`response to the mouse V regions can still occur (LoBuglio
`et al.. Proc. Natl. Acad. Sci. USA 84. 4220-4224. 1989).
`The second method for humanizing mouse antibodies is
`more complicated but more extensively reduces the potential
`immunogenicity of the mouse antibody. In this method. the
`complementarity determining regions (CDRs) from the V
`regions of the mouse antibody are grafted into human V
`regions to create "reshaped" human V regions. These
`15 reshaped human V regions are then joillled to human C
`regions. The only portions of the final reshaped human
`antibody derived from non-human protein sequences are the
`CDRs. CDRs consist of highly variable protein sequences.
`They do not show species-specific sequences. For these
`20 reasons. a reshaped human antibody carrying murine CDRs
`should not be any more immunogenic thalll a natural human
`antibody containing human CDRs.
`As seen from the above. it is supposed that reshaped
`human antibodies are useful for therapeutic purposes. but
`25 reshaped human antibodies to the human IL-6R are not
`known. Moreover. there is no process for construction of a
`reshaped human antibodies. universally applicable to any
`particular antibody. Therefore to construct a fully active
`reshaped human antibody to a particular antigen. various
`30 devices are necessary. Even though mouse monoclonal
`antibodies to the human Il--6R. i.e .. PMl and MT18. were
`prepared (Japanese Patent AWlication N<>. 2-189420). and
`the present inventors prepared mouse monoclonal antibodies
`to the human IL-6R. i.e .. AUK12-20. AUK64-7 and
`35 AUK146-15. the present inventors are not aware of publi(cid:173)
`cations which suggest construction of reshaped human anti(cid:173)
`bodies to the human IL-6R.
`The present inventors also found that. when the mouse
`monoclonal antibodies to the human D....-6R were injected
`into nude mice transplanted with a human myeloma cell line.
`the growth of the tumor was remarkably inhibited. This
`suggests that the anti-human IL-6 receptor antibody is useful
`as a therapeutic agent for the treatment of myeloma
`DISCT.OSURE OF INVENTION
`Therefore. the present invention is intemded to provide a
`less immunogenic antibody to the human IL-6R.
`Accordingly. the present invention provides reshaped human
`antibodies to the human IL-6R. The present invention also
`provides human/mouse chimeric antibodies useful during
`the construction of the reshaped human antibody. The
`prese.nt invention further provides a part of reshaped human
`antibody. as well as the expression systems for production of
`the reshaped human antibody and a part thereof. and of the
`55 chimeric antibody.
`More specifically. the present invention provides L chain
`V region of mouse monoclonal antibody to the human
`IL-6R; and H chain V region of a mouse monoclonal
`antibody to the human IL-6R.
`The present invention also provides a Chimeric antibody
`to the human Il..-6R. comprising:
`(1) an L chain comprising a human L chain C region and
`an L chain V region of a mouse monoclonal antibody
`to the Il..-6R; and
`(2) an H chain comprising a human H chain C region and
`an H chain V region of a mouse monoclonal antibody
`to the human Il..-6R.
`
`40
`
`BACKGROUND ART
`Intecleukio-6 (Il..-6) is a multi-function cytokine that is
`produced by a range of cells. It regulates immune responses.
`acute phase reactions. and hematopoiesis. and may play a
`central role in host defense mechanisms. It acts on a wide
`range of tissues. exerting growth-inducing. growth
`inhibitory. and differentiation-inducing effects. depending
`on the nature of the target cells. The specific receptor for
`IL-6 (Il..-6R) is expressed on lymphoid as well as non(cid:173)
`lymphoid cells in accordance with the multifunctional prop(cid:173)
`erties of Il..-6. Abnormal expression of the IL-6 gene has
`been suggested to be involved in the pathogenesis of a
`variety of diseases. especially autoimmune diseases. mesan(cid:173)
`gial proliferative glomeruJonephritis. and plasmacytoma/
`myeloma (see review by Hirano et al .. Immunol. Today 11.
`443-449. 1990). Human myeloma cells are observed to
`produce IL-6 and express Il..-6R. In experiments. antibody
`against IL-6 inhibited the in vitro growth of myeloma cells
`thus indicating that an autocrine regulatory loop is operating
`in oncogenesis of human myelomas (Kawano et al .. Nature.
`332. 83. 1988).
`The IL-6R is present on the surface of various animal 45
`cells. and specifically binds to IL-6. and the number of
`IL-6R molecules on the cell surface has been reported (Taga
`et al .. J. Exp. Med. 196. 967. 1987). Further, cDNA coding
`for a human Il..-6R was cloned and a primary structure of the
`IL-6R was reported (Yamasaki et al .. Science. 241. 825. 50
`1988).
`Mouse antibodies are highly immunogenic in humans
`and. for this reason. their therapeutic value in humans is
`limited. The half-life of mouse antibodies in vivo in human
`is relatively short. In addition. mouse antibodies can not be
`administered in multiple doses without generating an
`immune response which not only interferes with the planned
`efficacy but also risks an adverse allergic response in the
`patient.
`To resolve these problems methods of producing human- ro
`ized mouse antibodies were developed. Mouse antibodies
`can be humaniz.ed in two ways. The more simple method is
`to construct chimeric antibodies where the V regions are
`derived from the original mouse monoclonal antibody and
`the C regions are derived from suitable human antibodies. 65
`The resulting chimeric antibody contains the entire V
`domains of the original mouse antibody and can be expected
`
`26 of 95
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`5.795.965
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`3
`The present invention also provides CDR of an L chain V
`region of a mouse monoclonal antibody to the human IL-6R:
`and CDR of an H chain V region of a mouse monoclonal
`antibody to the human ll.-6R.
`The present invention moreover provides a reshaped
`human L chain V region of an antibody to the human IL-6R.
`comprising:
`(1) frameworkregions(FRs) of a human L chain V region.
`and
`(2) CDRs of an L chain V region of a mouse monoclonal
`antibody to the human IL-6R; and
`a reshaped human H chain V region of an antibody to
`the human Il.-6R comprising:
`(1) FRs of a human H chain V region. and
`(2) CDRs of an H chain V region of a mouse
`monoclonal antibody to the human Il.-6R.
`The present invention also provides a reshaped human L
`chain of an antibody to the human IL-6R. comprising:
`(1) a human L chain C region; and
`(2) an L chain V region comprising human FRs. and
`CDRs of a mouse monoclonal antibody to the human
`IL-6R; and
`a reshaped human H chain of an antibody to the human
`IL-6R. comprising:
`(1) a human H chain C region. and
`(2) an U chain V region comprising a human FRs.
`and CDRs of a mouse monoclonal antibody to the
`human Il.-6R.
`The present invention still more provides a reshaped 30
`human antibody to the human IL-6R. comprising:
`(A) an L chain comprising.
`(1) a human L chain C region. and
`(2) an L chain V region comprising human L chain FRs,
`and L chain CDRs of a mouse monoclonal antibody 35
`to the human Il.-6R; and
`(B) an H chain comprising.
`(1) a human H chain C region. and
`(2) an H chain V region comprising human H chain
`FRs. and H chain CDRs of a mouse monoclonal. 40
`antibody to the human IL-6R.
`The present invention further provides DNA coding for
`any one of the above-mentioned antibody polypeptides or
`parts thereof.
`The present invention also provides vectors. for example. 45
`expression vectors comprising said DNA.
`The present .invention furthe.r provides host cells trans(cid:173)
`formed or transfectcd with the said vector.
`The present invention still moce provide a process for
`production of a chimeric antibody to the human IL-6R. and so
`a process for production of reshaped human antibody to the
`human Il.-6R.
`
`4
`FIG. 5 is a graph showing a result of ELISA testing the
`ability of the present chimeric antibodies PM la and PMJb to
`inhibit ll.-6 from binding to the human Il..-6R.
`FIG. 6 is a diagram of the conslruction of the first version
`s of a reshaped human PM-1 H cha.in V region.
`FIG. 7 is a diagram of the construction of the fust version
`of a reshaped human PM-1 L chain V region.
`FIG. 8 represents a process for construction of an expres-
`10 sion plasmid HEF- 12h-gyl comprising a human elongation
`factor la (HEF-la) promoter/enhancer. useful for the
`expression of an H chain.
`FIG. 9 represents a process for construction of an expres(cid:173)
`sion plasmid HEF-12k-gk comprising the HEF- la
`is promoter/enhancer system. useful for the expression of an L
`chain.
`FIG. 10 represents a process for construction of an
`expression plasmid DHFR-PMh-gyl comprising HCMV
`promoter/enhancer and the dihydrofolate. reductase ( dhfr)
`20 gene linked to a defective SV40 promoter/enhancer
`sequence for amplification. useful for expression of an H
`chain.
`FIG. 11 represents a process for the construction of an
`expression plasmid DHFR-AE-RVh-PM 1-f comprising EFla
`2S promoter/enhancer and dhfr gene linked to a defective SV40
`promoter/enhancer sequence for amplification. useful for
`expressio~ of an H chain.
`FIG. U is a graph showing an ability of version "a" and
`"b" of the reshaped human PM- 1 L chain V region for
`binding to the human IL--6R.
`FIG. 13 is a graph showing an ability of version "f" of the
`reshaped human PM-1 H chain V region plus version "a" of
`the reshaped PM-1 L chain L chain V region for binding to
`the human llr6R.
`FIG. 14 is a graph showing an ability of vergion "f" of the
`reshaped PM-I H chain V region plus version "a" of the
`reshaped PM-1 L chain V region to inhibit the binding of
`IL-6 to the human Il.--6R.
`FIG. 15 represents expression plasmids HEF-V cgk and
`HEF-V H"gyl comprising a human EFI -a promoter/
`enhancer. useful for expression of an L chain and H chain
`respectively.
`FIG. 16 shows a process for construction of DNA coding
`for reshaped human AUK 12-20 antibody L chain V region.
`FIG. 17 is a graph showing results of an ELISA for
`confirm of an ability of a reshaped human AUK 12-20
`antibody L chain V region to bind to human ll.-6R. In the
`Figure. "Standard AUK 12-20 (chimera) means a result for
`chimeric AUK 12-20 anubody produced by CHO cells and
`purified in a large amount.
`FIG. 18 is a graph showing a result of an ELISA for an
`ability of a reshaped human AUK 12-20 antibody (L chain
`version "a"+H chain version "b") to bind to human IL-6R.
`FIG. 19 is a graph showing a result of an ELISA for an
`ability of a reshaped human AUK 12-20 antibody (L cha.in
`version "'a"+H chain version "'d") to bind to the human
`IL-6R.
`FIG. 2t shows a process for chemical synthesis of a
`reshaped human sle 1220 H anul>ody H cha.in V region.
`FIG. 21 is a graph showing a result of an ELISA for an
`ability of a reshaped human sle 1220 antibody (L chain
`version "a"+H cha.in version "'a") to .inlubit the binding of
`65 IL-6 to the human Il.-6R.
`FIG. 22 is a graph showing a result of an ELISA for an
`ability of a reshaped human sle 1220 antibody (L chain
`
`SS
`
`BRIEF DESCRIPTION OF DRAWINGS
`FIG. 1 represents expression vectors comprising human
`cytomegalo virus (HCMV) promoter/enhancer system. use(cid:173)
`ful for the expression of the present antibody peptide.
`FIG. 2 is a graph showing a result of ELISA for confir(cid:173)
`mation of an ability of the present chimeric antibody 60
`AUK12-20 to bind to the human IL-6R.
`FIG. 3 is a graph showing a result of measurement of an
`ability of the present chimeric antlOody AUK12-20 to inhibit
`the binding of Ilr6 to the human IL-6R.
`FIG. 4 .is a graph showing a result of ELISA for binding
`of the present chimeric antibodies PMla and PMlb to
`human IL-6R.
`
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`

`5
`version "a"+H chain version "b") to inhibit the binding of
`Il.-6 to the human Il..-6R.
`FIG. 23 is a graph showing a result of an ELISA for an
`ability of a reshaped human sle 1220 antibody (L chain
`version "a"+H chain version .. c") to inhibit the binding of
`Il.-6 to the human Il..-6R.
`FIG. 24 is a graph showing a result of an ELISA for an
`ability of a reshaped human sle 1220 antibody (L chain
`version "a"+H chain version ''d") inhibit the binding of D..,-6
`to the human U-6R.
`
`10
`
`5.795.965
`
`6
`cleaved pUC19 to obtain a plasmid incorporating a DNA
`fragment coding for a desired V region of a mouse mono(cid:173)
`clonal antibody.
`The sequencing of the cloned DNA can be carried out by
`s any conventional procedure.
`The cloning of the desired DNA. and the sequencing
`thereof. are described in detail in Examples J to 3.
`
`Best Mode for Carrying O\Jt the Invention Cloning
`of DNA codi.og for mouse V regions
`More specifically. to clone DNA coding for V regions of
`a mouse monoclonal antibody to a human Il.-6R. the con(cid:173)
`struction of hybridoma. which produces a monoclonal anti(cid:173)
`body to the human II...-6R. is necessary as a gene source. As
`such a hybrldoma. Japanese Patent Application No.
`2- 189420 describes a mouse hybridoma PM-1 which pro(cid:173)
`duces a monoclonal antibody PM 1 and the properties
`thereof. Reference Examples 1 and 2 of the present speci(cid:173)
`fication describe the construction process of the hybridoma
`PM 1. The present inventors have constructed hybridomas
`AUK12-20. AUK64-7. and AUK146-15. each producing a
`mouse monoclonal antibody to the human JL.6R. The con(cid:173)
`struction process of these hybridomas is desaibed in the
`Reference Examples 3 of this specification.
`To clone desired DNAs coding for V regions. of a mouse
`monoclonal antibody. hybridoma cells are homogenized and
`a total RNA is obtained according to a conventional proce(cid:173)
`dure described by Chirgwin et al .. Biochemistry 18. 5294.
`1977. Next. the total RNA is used to synthesize siogle(cid:173)
`stranded cDNAs according to the method descri.bed by J. W.
`Larrick et al.. Biotechnology. 7. 934. 1989.
`Next. a specific amplification of a relevant portion of the
`cDNA is carried out by a polymerase chain reaction (PCR)
`method- For amplification of a IC L chain V region of a mouse
`monoclonal antibody. 11 groups of oligonucleotide primers
`(Mouse Kappa Variable; MKV) represented in SEQ ID NO:
`1 to 11. and an oligooucleotide primer (Mouse Kappa
`Constant; MKC) represented in SEQ ID NO: 12 are used as
`5'-tenninaJ primers and a 3'-terminal primer respectively.
`The MKV primers hybridize with the DNA sequence coding
`for the mouse lC L chain leader sequence. and the MKC
`primer hybridizes with the DNA sequence codi.og foe the
`mouse IC L chain constant region. For amplification of the H
`chain V region of a mouse rnonoclonal anlll>ody. 10 groups
`of oligonucleotide primers (Mouse Heavy Variable; MHV)
`represeoted in SEQ ID NO: 13 to 22. and a oligonucleotide
`primer (Mouse Heavy Constant MHC) represented in SEQ
`ID NO: 23 are used as 5'-terminal primers and a 3'-terminal
`primer. respectively.
`Note. the 5'-terminal primers contain the nucleotide
`sequence GrCGAC near the 5'-end thereof. which sequence
`provides a restriction enzyme Sal I cleavage site; and the
`3'-terroinaJ primer contai.ns the nucleotide sequence
`CCCGGG near the 5-end thereof. which sequence provides
`a restriction enzyme Xma I cleavage site. These restriction
`enzyme cleavage sites are used to subclone the DNA frag(cid:173)
`ments coding for a variable region into cloning vectors.
`Next. the amplification product is cleaved with restriction
`enzymes Sal I and Xma I to obtain a DNA fragment coding
`for a desired V region of a mouse monoclonal antibody. On
`the other hand. an appropriate cloning vector such as plas(cid:173)
`mid pU09 is cleaved with the same restriction enzymes Sal
`I and Xma I and the above DNA fragment is ligated with the
`
`Complementarity Determining Regions (CDRs)
`The present invention provides hypervariable or comple-
`mentari ty determining regions (CDRs) of each V region of
`the present invention. The V domains of each pair of L and
`H chains from the antigen binding site. The domains on the
`is L and H chains have the same general structure and each
`domain comprises four framework regions (FRs). whose
`sequences are relatively conserved. connected by three
`CDRs (see Kabat. E. A .. Wu. T. T .. Bil of sky. H .. Reid-Miller.
`M . and Perry. H .. in "Sequences of Proteins of Immuno-
`20 logical Interest". US Dept Health and Human Services
`1983). The four FRs largely adopt a ~sheet conformation
`and the CDRs form loops connecting FRs. and in some cases
`fooning part of. the ~sheet structure. The CDRs are held in
`close proximity by FRs and. with the CDRs from the other
`domain. contribute to the formation of the antigen binding
`site. The CDRs are described in Example 4.
`
`25
`
`4S
`
`Construction of Chimeric Antibody
`Prior to designing reshaped human V reg.ions of an
`30 antibody to the human IL-6R. it is necessary to confirm that
`the CDRs to be used actually form an effective antigen
`binding region. Fa this purpose. chimeric antibodies were
`constructed. In addition the amino acid sequences of V
`regions of mouse anti human IL-6R antibodies predicted
`35 from the nucleotide sequences of cloned DNAs of the 4
`mouse monoclonal anlibodies desaibed in Ex.ample 1 and 2
`were compared to each other and to V regions from known
`mouse and bum.an antibodies. For each of the 4 mouse
`monoclonal antibodies. a set of typical. functional mouse L
`40 and H chain V regions had been cloned. All four mouse
`anti-IL-6R anllbodies. however. bad relatively distinct V
`regions. The 4 antibodies were not simply minor variations
`of each other. Using the cloned mouse V regions. 4 chimeric
`anti-Il.-6R antibodies were constructed.
`The basic method fa constructing chimeric anl!l>od.ies
`comprises joining the mouse leader and V region sequences.
`as found in the PCR--cloned cDNAs. to hwnan C regions(cid:173)
`coding sequence already present in mammalian cell e.xpres(cid:173)
`sioo vectors. Among said 4 monoclonal antibodies. con-
`so struction of a chimeric antibody from the monoclonal
`antibody AUK12-20 is desaibed in Example 5.
`Construction of a chimeric antibody from the monoclonal
`antibody PM-1 is described in Example 6. The cDN A codi.og
`for the mouse PM- 1 lC L chain leader and V region was
`ss PCR-subcloned into an exp-ession vector containing a
`genomic DNA coding foe the human kappa C region. The
`cD