.
`-·---- ---
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`
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`
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`
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`
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`
`DECEMBER 1989
`VOLUME 86
`NUMBER 24
`
`Proceedings
`
`OF THE
`
`National Acaden1y
`of Sciences
`
`OF THE UNITED STATES OF AMERICA
`
`BIOEPIS EX. 1034
`Page 1
`
`

`

`Proceedings
`OF THE
`National Academy
`of Sciences
`OF THE UNITED STATES OF AMERICA
`
`Officers
`of the
`Academy
`
`Editorial Board
`of the
`Proceedin~:s
`
`FRANK PRESS, President
`JAMES D. EBERr, Vice President
`PETER H. RAVEN, 1/ome Secretary
`WILLIAM E. GORDON, Ford~:n Secretary
`ELKAN R. BLOUT, Trea.\'1/rer
`
`RoBERT H. ABELES
`GoRDON A. BA YM
`RoNALD BRESLOW
`MICHAEL J. CHAMBERLIN
`MARY-DELL CfiiLTON
`
`IGOR U. DAWID, Chairman
`EDWARD E. DAVID, JR.
`RONALD l.GRAIIAM
`STUART A. KORNFELD
`DANIEL E. Kosm.AND, JR.
`I'IIILII' w. MAJERUS
`
`DANIEL NATIIANS
`MAXINE F. SINGER
`SOLOMON H. SNYDER
`HAROLD V ARM US
`THOMAS A. WALDMANN
`
`Mana~:in~: Editor: FRANCES R. ZwANZIG
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`
`BIOEPIS EX. 1034
`Page 2
`
`

`

`Proc. Nat/. Acad. Sci. USA
`Vol. 86, pp. 10029-10033, December 1989
`Immunology
`
`A humanized antibody that binds to the interleukin 2 receptor
`(chimeric antibody I antibody affinity I autoimmune disease)
`
`CARY QuEEN*, WILLIAM P. ScHNEIDER*, HAROLD E. SELICK*i·, PHILIP W. PAYNE*,
`NICHOLAS F. LANDOLFI*, JAMES F. DuNCAN*t, NEVENKA M. AvDALOVIc*, MICHAEL LEVITT§,
`RICHARD P. JUNGHANS~!, AND THOMAS A. WALDMANN~
`*Protein Design Labs, 3181 Porter Drive. Palo Alto, CA 94304; §Department of Cell Biology, Stanford University, Stanford, CA 94305; and ~Metabolism
`Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
`
`Contribwed by Thomas A. Waldmann, August 30, 1989
`
`The anti-Tac monoclonal antibody is known
`ABSTRACT
`to bind to the pSS chain of the human intcrlcukin 2 receptor and
`to inhibit proliferation of T cells by blocking interleukin 2
`binding. However, usc of anti-Tac as an immunosuppressant
`drug would be impaired by the human immune response
`against this murine antibody. We have therefore constructed a
`"humanized" antibody by combining the complementarity(cid:173)
`determining regions (CDRs) of the anti-Tac antibody with
`human framework and constant regions. The human frame(cid:173)
`work regions were chosen to maximize homology with the
`anti-Tac antibody sequence. In addition, a computer model of
`murine anti-Tac was used to identify several amino acids
`which, while outside the CDRs, arc likely to interact with the
`CDRs or antigen. These mouse amino acids were also retained
`in the humanized antibody. The humanized anti-Tac antibody
`has an affinity for pSS of3 X 109 M- 1, about 113 that of murine
`anti-Tac.
`
`The cellular receptor for the lymphokine interleukin 2 (IL-2)
`plays an important role in regulation of the immune response
`(reviewed in ref. 1). The complete IL-2 receptor (IL-2R)
`consists of at least two IL-2-binding peptide chains: the p55
`or Tac peptide (2, 3), and the recently discovered p75 peptide
`(4, 5). Identification and characterization of the p55 peptide
`were facilitated by the development of a monoclonal anti(cid:173)
`body, anti-Tac, which binds to human p55 (2). The p55
`peptide was found to be expressed on the surface ofT cells
`activated by an antigen or mitogen but not on resting T cells.
`Treatment of human T cells with anti-Tac antibody strongly
`inhibits their proliferative response to antigen or to IL-2 by
`preventing IL-2 binding (3, 6).
`These results suggested that anti-IL-2R antibodies would
`be immunosuppressive when administered in vivo. Indeed,
`injection of an anti-IL-2R antibody into mice and rats greatly
`prolonged survival of heart allografts (7, 8). Anti-IL-2R was
`also effective in rats against experimental graft-versus-host
`disease (9). In animal models of autoimmune disease, an
`anti-IL-2R antibody alleviated insulitis in nonobese diabetic
`!llice and lupus nephritis in NZB x NZW mice (10). Anti-Tac
`Itself was highly effective in prolonging survival of kidney
`allografts in cynomolgus monkeys (11).
`In human patients, the specificity of anti-Tac for activated
`T cells might give it an advantage as an immunosuppressive
`agent over OKT3 (monoclonal anti-CD3), which is effective
`in treating kidney transplant rejection (12), but which sup(cid:173)
`presses the entire peripheral T-cell population. In fact, in
`phase I clinical trials for kidney transplantation, prophylactic
`a~ ministration of anti-Tac significantly reduced the incidence
`ot early rejection episodes, without associated toxicity (13).
`Furthermore, treatment with anti-Tac induced temporary
`
`The publication cos Is of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked" advertisement"
`m accordance with l!l U.S.C. ~1734 solely to indicate this fact.
`
`partial or complete remission in three of nine patients with
`Tac-expressing adult T-ceii leukemia (14). However, as a
`murine monoclonal antibody, anti-Tac elicits a strong human
`antibody response against itself, as does OKT3 (15). This
`response would prevent its long-term use in treating autoim(cid:173)
`mune conditions or suppressing organ transplant rejection.
`The immune response against a murine monoclonal anti(cid:173)
`body may potentially be reduced by transforming it into a
`chimeric antibody. Such antibodies, produced by methods of
`genetic engineering, combine the variable (V) region binding
`domain of a mouse (or rat) antibody with human antibody
`constant (C) regions (16-18). Hence, a chimeric antibody
`retains the binding specificity of the original mouse antibody
`but contains less amino acid sequence foreign to the human
`immune system. Chimeric antibodies have been produced
`against a number of tumor-associated antigens (19-21). In
`some but not all cases, the chimeric antibodies have mediated
`human complement-dependent cytotoxicity (CDC) or anti(cid:173)
`body-dependent cellular cytotoxicity (ADCC) more efficient(cid:173)
`ly than the mouse antibodies (21).
`When the murine antibody OKT3 is used in human pa(cid:173)
`tients, much of the resulting antibody response is directed
`against the V region of OKT3 rather than the C region (15).
`Hence, chimeric antibodies in which the V region is still
`nonhuman may not have sufficient therapeutic advantages
`over mouse antibodies. To further reduce the immunogenic(cid:173)
`ity of murine antibodies, Winter and coiieagues constructed
`"humanized" antibodies in which only the minimum neces(cid:173)
`sary parts of the mouse antibody, the complementarity(cid:173)
`determining regions (CDRs), were combined with human V
`region frameworks and human C regions (22-25). We report
`here the construction of chimeric and humanized anti-Tac
`antibodies.!! For the humanized antibody, sequence homol(cid:173)
`ogy and molecular modeling were used to select a combina(cid:173)
`tion of mouse and human sequence elements that would
`reduce immunogenicity while retaining high binding affinity.
`
`MATERIALS AND METHODS
`Construction of Plasmids. eDNA cloning was by the
`meth?d of Gubler and Hoffman (26), and sequencing was by
`the dtdeoxy method (27). The plasmid pVKl (Fig. 1A) was
`constructed f:om the followin? fragments: an approximately
`4550-base-patr (bp) BamHI-EcoRI fragment from the plas-
`
`~bbreviati.o~s: IL-~R, interleukin 2 receptor; CDR, complementar(cid:173)
`Ity-determ~n.~ng region; CDC, complement-dependent cytotoxicity·
`~p~c. antibody-dependent cellular cytotoxicity; V, variable· /
`'
`JOining; C, constant.
`i"Present address: Biospan, 440 Chesapeake Drive Redwood c·t
`CA 94063.
`'
`I y,
`:f.Present address: Beckman Instruments, 1050 Page Mill p
`. d p 1
`'-0
`Alto, CA 94304.
`' a o
`<l
`liThe sequences reported in this paper have been d
`·
`·
`GenBank data base (accession nos. M28250 and M~~~~1{).d In the
`
`'
`
`BIOEPIS EX. 1034
`Page 3
`
`

`

`10030
`
`Immunology: Queen ct a/.
`
`Proc. Nat/. Accul. Sci. USA 86 ( 1989)
`
`A
`
`EH
`
`Eco AI
`
`Eco AI
`
`~l.\ VJ
`
`Xba I
`
`mouse genomic DNA and therefore includes a splice donor
`signal. The finallO nucleotides of the oligonucleotide include
`an Xba I site.
`We hybridized this oligonucleotide to M13L and extended
`it with the Klenow fragment of DNA polymerase. The DNA
`was heat-denatured, hybridized with an excess of the "re(cid:173)
`verse primer" 5'-AACAGCTATGACCATG-3', again ex(cid:173)
`tended with Klenow DNA polymerase, and digested with
`Xba I. The digested DNA was run on a gel, and an approx(cid:173)
`imately 400-bp fragment was excised and inserted into the
`Xba I site of p V Kl. Sequencing showed that the fragment
`consisted of the V-J region of the light chain eDNA followed
`by the splice donor "tail," as expected (Fig. lB), and pLTac,
`a clone with the appropriate orientation, was chosen. In an
`analogous fashion, the heavy chain V-J segment, followed by
`the mouse J 112 splice donor sequence, was excised from
`Ml3H and inserted into the Xba I site ofp V y1 to yield pGTac.
`Computer Analysis. Sequences were manipulated and ho(cid:173)
`mology searches were performed with the MicroGenie Se(cid:173)
`quence Analysis Software (Beckman). The molecular model
`of the anti-Tac V region was constructed with the ENCAD
`program (35) and examined with the MIDAS program <36) on
`an IRIS 40-120 graphics workstation (Silicon Graphics).
`Construction of Genes for Humanized Antibody. Nucleotide
`sequences were selected that encoded the protein sequences
`of the humanized light and heavy chain V regions including
`signal peptides (Results), generally utilizing codons found in
`the mouse anti-Tac sequence. These nucleotide sequences
`also included the same splice donor signals used in the
`chimeric genes and an Xha I site at each end. For the heavy
`chain V region, four overlapping 120- to 130-nucleotide-long
`oligonucleotides were synthesized that encompassed the
`entire sequence on alternating strands. The oligonucleotides
`were phosphorylated with polynucleotide kinase, annealed,
`extended with T4 DNA polymerase, cut with Xba I, and
`ligated into the Xba I site of pUC19 <34), using standard
`reaction conditions. An insert with the correct sequence was
`recloned in pVyl. The humanized light chain V region was
`constructed similarly.
`Transfections. For each antibody constructed, the light
`chain plasmid was first transfected into Sp2/0 mouse my(cid:173)
`eloma cells (AT'I'C CRL 1581) by electroporation (Bio-Rad
`Gene Pulser) and cells were selected for RPf expression (28).
`Clones secreting a maximal amount of light chain, as deter(cid:173)
`mined by ELISA, were transfected with the heavy chain
`plasmid and cells were selected for hygromycin B resistance
`(31). Clones secreting a maximal amount of complete anti(cid:173)
`body were detected by ELISA. The clones were used for
`preparation or chimeric and humanized antibodies.
`Antibody Purification. Medium from confluent cells was
`passed over a column of staphylococcal protein A-Sepharose
`CL-48 (Pharmacia), and antibody was eluted with 3 M
`MgCiz. Antibody was further purified by ion-exchange chro(cid:173)
`matography on Baker Bond ABx (J. T. Baker). Final anti(cid:173)
`body concentration was determined, assuming that 1 mg/ml
`has an AzKo of 1.4. Anti-Tac antibody itself was purified as
`described (2).
`Affinity Measurements. Affinities were determined by com(cid:173)
`petition binding. HuT-102 human T-lymphoma cells (ATTC
`TIB 162) were used as source of p55 Tac antigen. Increasing
`amounts of competitor antibody (anti-Tac, chimeric, or hu(cid:173)
`manized) were added to 1.5 ng of radioiodinated (Pierce
`Iodo-Beads) tracer anti-Tac antibody (2 11-Ci/ 11-g; 1 Ci = 37
`GBq) and incubated with 4 x 105 HuT cells in 0.2 ml of
`binding buffer (RPMI 1040 medium with 10% fetal calf serum,
`human IgG at 100 11-g/ml, 0.1% sodium azide) for 3 hr at room
`temperature. Cells were washed and pelleted, and their
`radioactivities were measured, and the concentrations of
`bound and free tracer antibody were calculated. The affinity
`of mouse anti-Tac was determined by Scatchard plot analy-
`
`Gpt
`
`Hybridize
`primer
`
`v
`Extend
`
`8
`
`Xba I
`
`l
`Denature. l
`l
`
`'f-'o3\
`
`,.3/
`c
`J
`
`~
`
`Hybridize
`rev. primer ~
`--~------------
`
`Extend
`and cut
`
`Xba I
`
`v
`
`FIG. 1.
`(A) Schematic diagram of the plasmids pVKl and pLTac.
`Light chain exons are shown as boxes. An arrow indicates the
`direction of transcription from the K promoter. E11, heavy chain
`enhancer. Not drawn to scale. (B) Schematic diagram of the method
`used to excise the V-1 region. SD, splice donor sequence: rev.
`primer, reverse primer.
`
`mid pSV2gpt (28) containing the amp and RfJl genes; an
`1800-bp EcoRI-B!;l II fragment from pKcatH (29) containing
`the heavy chain enhancer and K promoter; and a 1500-bp
`EcoRI-Xha I fragment containing the human CK region (30).
`Similarly, pVy1 was constructed starting from a 4850-bp
`BamHI-EcoRI fragment of the plasmid pSV2hph (a gift of A.
`Smith, A. Miyajima, and D. Strehlow, Stanford University),
`which is analogous to pSV2gpt except that the RPf gene is
`replaced by the hyR gene (31). This fragment was combined
`with the EcoRI-B!;I II fragment from pKcatH and a 2800-bp
`Hindlli-Pvu II fragment containing the human y1 constant
`region, isolated from a phage kindly provided by L. Hood
`(32). In each case, the fragments were combined by standard
`methods (ref. 33, pp. 390-401), with an Xha I linker inserted
`between the K promoter fragment and the 5' end of the C
`region fragment.
`Construction of Chimeric Genes. EcoRI fragments contain(cid:173)
`ing the anti-Tac light and heavy chain cDNAs were sepa(cid:173)
`rately inserted into the EcoRI site of the phage Ml3mpllD,
`a variant of M13mpll (34) in which the EcoRI and Xha I sites
`of the polylinker were filled in and joined. The resulting
`phage, in which the 5' ends of the cDNAs abutted the Xba I
`site, were respectively denoted Ml3L and M13H. The V-J (J,
`joining) segments of the cDNAs, followed by splice donor
`signals, were precisely excised from these phage, using a
`double-priming scheme <Fig. 1B). For the light chain, , ~1e
`following primer was synthesized (Applied Biosyster·;s
`model 3808 DNA synthesizer) and purified by gel electro(cid:173)
`phoresis: 5'-CCAGAATTCTAGAAAAGTGTACTTAC(cid:173)
`GTTTCAGCTCCAGCTTGGTCCC-3'. From the 3' end, the
`first 22 residues of the primer are the same as the last 22
`residues of the JK5 segment (noncoding strand). The next 16
`nucleotides are the same as the sequence that follows J K5 in
`
`BIOEPIS EX. 1034
`Page 4
`
`

`

`Immunology: Queen et a/.
`
`Proc. Nat/. A cad. Sci. USA 86 ( 1989)
`
`10031
`
`sis, using anti-Tac itself as the competitor. Then the affinities
`of the chimeric and humanized antibodies were each calcu(cid:173)
`[anti-Tac] = (1/ Kx) -
`lated according to the formula [X] -
`(1/ Ka), where Ka is the affinity of anti-Tac (9 x 109 M-'), Kx
`is the affinity of the competitor X, [ ] indicates the concen(cid:173)
`tration of competitor antibody at which bound/free tracer
`binding is R0/2, and R0 is maximal bound/free tracer binding
`(37).
`
`RESULTS
`Cloning of Light and Heavy Chain eDNA. A eDNA library
`in AgtlO was prepared from anti-Tac hybridoma cells and
`screened with oligonucleotide probes for the mouse K and y2a
`constant regions. The eDNA inserts from four K-positive and
`four y2a-positive phage were subcloned in M13mp19. Partial
`sequencing showed that two of the K isolates had one
`sequence, and the other two had another sequence. In one
`pair, a V K gene segment was joined to the JK2 segment out of
`its reading frame. In addition, the conserved cysteine at
`position 23 was absent from this V segment, and the se(cid:173)
`quences of the two isolates differed slightly. Presumably,
`these clones were the result of an aberrant joining event in
`one K allele, which continued to undergo somatic mutation
`after the formation of the hybridoma.
`The V-J segments of the other pair of K clones were
`sequenced completely and were identical. This light chain
`uses the 1 K5 segment. Partial sequencing of the four y2a
`clones showed they were all from the same gene. The V-J
`segments of two were sequenced completely and were iden(cid:173)
`tical. This heavy chain uses the JH2 segment and is of
`subgroup II (38). The DNA sequences have been deposited
`with GenBank; II the deduced protein sequences are shown in
`Fig. 2. As both alleles of the K light chain were accounted for
`and only one heavy chain sequence was detected, we tenta(cid:173)
`tively assigned these sequences to the anti-Tac antibody
`genes.
`Construction of Chimeric Genes. Plasmid vectors were
`prepared for the construction and expression of chimeric light
`and heavy chain genes. The plasmid pVKl (Fig. lA) contains
`the human genomic CK segment, including 336 bp of the
`preceding intron and the poly(A) signal. It also contains the
`promoter sequence from the MOPC 41 K gene and the heavy
`chain enhancer sequence, which synergize to form a very
`strong transcriptional unit (29). There is a unique Xba I site
`between the promoter and the intron. A similar plasmid,
`pVyl, was prepared by using the human Cyl region in place
`of the CK region. In that case, the region inserted between the
`Xba I and BamHI sites extended from about 210 bp 5' of the
`C11 1 exon to beyond the C113 exon.
`Our strategy was to insert the V-J region from the anti-Tac
`K eDNA, followed by a splice donor signal, at the Xba I site
`
`A
`
`B
`
`of p V Kl to construct the plasmid pL Tac. Doing so created a
`chimeric K gene with a short synthetic intron between the
`mouse V-J and human CK segments (Fig. lA). For this
`purpose, we used a form of double primer-directed mutagen(cid:173)
`esis (Materials and Methods; Fig. lB). Similarly, the V-J
`region from the anti-Tac y2a heavy chain eDNA, followed by
`a splice donor signal, was inserted into the Xba I site ofp V yl.
`The resulting plasmid, pGTac, contained a chimeric heavy
`chain gene, with a synthetic intron between the mouse V-J
`and human Cyl segments.
`Construction of a Humanized Anti-Tac Antibody. In select(cid:173)
`ing a human antibody to provide the variable region frame(cid:173)
`work for the humanized anti-Tac antibody, we reasoned that
`the more homologous the human antibody was to the original
`anti-Tac antibody, the less likely would combining the anti(cid:173)
`Tac CDRs with the human framework be to introduce dis(cid:173)
`tortions into the CDRs. The anti-Tac heavy chain sequence
`was therefore compared by computer with all the human
`heavy chain sequences in the National Biomedical Research
`Foundation Protein Identification Resource (release 15). The
`heavy chain V region of the Eu antibody (of human heavy
`chain subgroup I; ref. 38) was 57% identical to the anti-Tac
`heavy chain V region (Fig. 2B); all other complete V 11 regions
`in the data bank were 30-52% identical. However, no one
`human light chain V region was especially homologous to the
`anti-Tac light chain. We therefore chose to use the Eu light
`chain (of human light chain subgroup I; ref. 38) together with
`the Eu heavy chain to supply the framework sequences for
`the humanized antibody. The CDRs in the humanized anti(cid:173)
`body were of course chosen to be identical to the anti-Tac
`CDRs (Fig. 2).
`.
`A computer program was used to construct a plausible
`molecular model of the anti-Tac V domain (Fig. 3), based on
`homology to other antibody V domains with. know~ cry~tal
`structure and on energy minimization. Graphic mampulation
`shows that a number of amino acid residues outside of the
`CDRs are in fact close enough to them to either influence
`their conformation or interact directly with antigen. When
`these residues differ between the anti-Tac and Eu antibodies,
`the residue in the humanized antibody was chosen to be the
`anti-Tac residue rather than the Eu residue. This choice was
`made for residues 27, 30, 48, 67, 68, 98, and 106 in the
`humanized heavy chain, and for 47 and 59 in the hum~miz~d
`light chain (Figs. 2 and 3; amino acids shown in blue m Fig.
`3), although we now consider the light chain residue 59,
`which was chosen on the basis of an earlier model, to be
`doubtful. In this way, we hoped to better preserve the precise
`structure of the CDRs at the cost of possibly making the
`humanized antibody slightly less "human."
`Different human light or heavy chain V regions exhibit
`strong amino acid homology outside of the CDRs, within the
`framework regions. However, a given V region will usually
`
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`I
`I
`P G Q G L E W ,!. G Y T N I' S T G Y T E Y
`
`61 R F I G S G S G I E F T L T I S S L Q !'
`I I
`I I I I I I
`I I I I
`60 R F ~ G S G S G T S Y S L T I S R H E A
`
`61 A Q K F Q G R V T
`I T A D E S T N T i\ Y
`I I I
`I
`I I I
`I I I
`I
`t5. f::. T L T A D K S S S T A Y
`61 N 0 Y F K ll
`
`81 D D F A T Y Y C Q Q Y N S D S K M F G Q
`I
`I I I I I
`I
`I I
`80 E D A A T Y Y G II 9 R S T Y P !. T F G S
`
`81 H E L S S L R S E D T A F Y F C A G G Y
`I
`I I I I
`I I
`I
`I
`I I
`I
`81 H Q L S S L T F E D S A Y, Y l: C A !':. Q_
`
`101 C T K V E V K
`I I I
`I
`I
`100 G T K L E L K
`
`101 G I Y S P E E Y N G G L V T V S S
`I
`I
`I I I I
`r, n V F D Y !:!, £ Q. G ! T L T V S S
`
`100
`
`FIG. 2. Amino acid sequences of the humanized
`anti-Tac light (A) and heavy (B) chains. The se(cid:173)
`quences of the Eu antibody light and heavy chains
`(upper lines) arc shown aligned above the mouse
`anti-Tac light and heavy chain sequences (lower
`lines), with a I indicating identity of amino acids.
`The three CDRs in each chain are underlined, and
`the other mouse amino acids used in the humanized
`antibody are double underlined. Hence, the human(cid:173)
`ized sequences are the same as the upper (Eu)
`sequences, except where the amino acid is umler(cid:173)
`lined or double underlined.
`
`BIOEPIS EX. 1034
`Page 5
`
`

`

`10032
`
`Immunology: Q uee n et a/.
`
`Proc. Na t/. A cad. Sci. USA 86 ( 1989)
`
`F I G . 3. Model of the mouse anti -Tac anti body Y region, generated with the EN CAD progra m and di ~ playcd w ilh th e M I DA S program. Am in o
`ac ids in the CDRs are show n in reel: amino acid s potent iall y interacting with th e CD Rs arc shown in blue: other mou se am in o ac ids used in th e
`humani zed antibody are shown in yellow, as descri bed in the tex t. Thu s, all am ino ac id s tran sferred from the anti -Tac seq uence to the hum ani zed
`antibody are shown in reel, blue, or yellow. Res idue 1 i ~ th e first amino ac id of Y K: residue 30 1 is the first am in o ac id of V 11 •
`
`contain excepti o na l ami no ac id s, atypica l o f oth er huma n V
`regio ns, at severa l framewo rk positions. T he E u a ntibody
`conta in s such unu sual re sidues at pos iti ons corre s po ndin g to
`93, 95, 98 , 106, 107, 108, a nd 110 or the huma ni zed heavy
`cha in a nd 47 a nd 62 of the li ght cha in (F ig. 2), as dete rmin ed
`by visua l compa ri so n of th e Eu heavy and lig ht cha in V
`regio ns w ith o th er huma n V regio ns o f subgro up I (38) . T he
`E u antibody co ntain s seve ra l oth e r unu sual residues , but at
`the listed pos itions , the murine a nti-T ac a ntibod y actually ha s
`a resi du e much more typica l of hum a n seq ue nces than does
`E u. At these positions , we therefore chose to use the a nti-Tac
`re sidue rather tha n the E u residue in th e humanized a ntibody,
`to make th e a ntibod y mo re gene ri call y huma n. So me of these
`res idu es had a lread y been se lected beca use of their prox imit y
`to th e CDR s, a s described above (th e rema ining o nes are
`s how n in ye llow in Fig. 3).
`These c rite ri a a ll owed the selec ti on of all am ino ac id s in the
`huma ni zed a ntibody V regio ns as co ming from e ith er anti-Tac
`o r E u (Fig. 2). DNA segme nt s e ncoding th e des ired heavy
`a nd li ght cha in a mino acid sequ e nces we re s ynthes ized .
`These DN A segme nt s a lso encoded typ ica l immunoglobulin
`sig na l seq ue nces fo r processing a nd secret io n , and they
`conta in ed s pli ce donor signa ls at their 3' end. T he light a nd
`heavy chain segme nt s were c loned , re specti ve ly , in pV Kl and
`pVyl to form the plas mid s pHuLTac a nd pHuGTac.
`Properties of C himeric and Humanized Antibodies. S p2/0
`ce ll s , a nonproducing mo use mye loma line , we re tran sfected
`seque nti a ll y w ith p L Tac and pGTac (c him e ri c ge nes) or w ith
`pHuL T ac a nd pHuGTac (humanized ge nes). Ce ll clones
`we re se lected fi rst fo r a ntibi oti c res istance and the n for
`maxima l a ntibody sec re ti on , w hi c h reac hed 3 ttg/ 106 cell s per
`24 hr . S l nucl ease ma pping o f RN A ext racted from th e cells
`tra nsfected w ith pLTac a nd pGTac s ho wed that the sy nth e tic
`in t ro ns between the V a nd C regio ns (Fig. 1A ) we re correctl y
`s pli ced (data not s how n). A nti body was purified fro m th e
`
`culture medium o f ce ll s prod uc ing th e c him eric or huma ni ze d
`an tibody. When a na lyzed by redu c ing SDS/ po lyac rylam ide
`ge l electrop ho resis , th e a nti bod ie s s howed o nl y two band s,
`hav ing th e expected molecu lar we ight s 50 ,000 and 25,000 .
`F low cy to metry s howed that th e c him e ri c and hum a ni zed
`a ntibodi es bound to Hut-102 a nd C RII .2 ce ll s, two huma n
`T-ce llline s th at exp re ss th e p55 c ha in of th e IL-2R . but no t
`to CEM a nd othe r ce ll line s th a t do no t e xpress th e I L-2 R. To
`determin e the binding affinit y of th e c him eric a nd humani zed
`antibodi es, th e ir ab ilit y to compe te with labe led mo use
`anti-Tac for binding to Hut-102 ce ll s wa s de te rmin ed. The
`affinit y of chim eric an ti -Tac wa s indi stingu is hab le fro m th a t
`of anti -Tac (dat a no t 'i how n) , as expec ted from th e fact th a t
`th eir e ntire V regio ns arc id e nti ca l. The a ffinit y of huma ni zed
`anti -Tac for me mbrane-bound p55 wa s 3 x 10~ M -
`I , abo ut
`1/3 th e meas ured affi nit y o f 9 X 10~ M - I o f a nti -Tac it se lf
`(Fig. 4).
`
`DISCUSSION
`
`Becau se mo noc lo na l antibodi e s ca n be produced th at a rc
`hi ghl y s pecifi c for a wid e vari e ty of ce llul a r targets, a ntibod y
`th e ra py ho ld s grea t prom ise for th e treatme nt of ca ncer,
`autoimmun e co nditi o ns , a nd o th e r di seases . H oweve r , thi s
`promise has no t been w id e ly rea lized , la rge ly because mo st
`monoclonal a nti bodi es, w hi ch are o f mo use o ri gin , a re im (cid:173)
`mun ogeni c when used in hum a n pati e nts a nd are in effecti ve
`at recruiting huma n immun e effector fun c ti o ns s uc h a s CDC
`and A DCC. A partial so luti o n to thi s prob le m is the use of
`c hime ric antibodie s 0 6) , w hi ch co mbine th e V region binding
`do ma in s of mou se antibod ies with huma n a ntibod y C regio ns .
`Initially , ch im e ri c a ntibodi es we re co nstructed by co mbining
`ge no mi c c lones of the V a nd C region ge ne s. Howeve r , thi s
`meth od is very time co ns uming beca use o f the diffi cult y of
`ge no mi c c lo ning , e specially from te trap loid hybrid omas .
`
`BIOEPIS EX. 1034
`Page 6
`
`

`

`Immunology: Queen et a!.
`
`Proc. Nat!. A cad. Sci. USA 86 ( 1989)
`
`10033
`
`2 .5 r - - - - - - - - - - - - - - - - ,
`
`a:
`w
`u
`<(
`a:
`1-
`w
`w
`a:
`
`u. --0 z
`6 0.5
`
`Ill
`
`LOG CONCENTRATION OF COMPETITOR, pM
`
`FIG. 4. Competitive binding of labeled anti-Tac tracer to Hut-102
`cells. Duplicate samples are shown. •. Mouse anti-Tac competitor;
`7, humanized anti-Tac competitor.
`
`More recently, eDNA clones of the V and C regions have
`been combined, but this method is also tedious because of the
`need to join the V and C regions precisely (20, 21). Here we
`show that the V region from a readily obtainable eDNA clone
`can be easily joined to a human genomic C region, which need
`only be cloned once, by leaving a synthetic intron between
`the V and C regions. When linked to suitable transcriptional
`regulatory elements and transfected into an appropriate host
`cell, such chimeric genes produce antibody at a high level.
`Chimeric antibodies represent an improvement over
`mouse antibodies for usc in human patients, because they are
`presumably less immunogenic and sometimes mediate CDC
`or ADCC more effectively (21). For example, chimeric
`anti-Tac mediates ADCC with activated human effector cells,
`whereas murine anti-Tac docs not (unpublished data). How(cid:173)
`ever, the mouse V region can itself be highly immunogenic
`(15). Winter and colleagues therefore took the further, inno(cid:173)
`vative, step of combining the CDRs from a mouse (or rat)
`antibody with the framework region from a human antibody
`(22-25), thus reducing the xenogeneic elements in the hu(cid:173)
`manized antibody to a minimum. Unfortunately, in some
`cases the humanized antibody had significantly less binding
`affinity for antigen than did the original mouse antibody. This
`is not