[CANCER RESEARCH 57. 4593—4599. October l5. I997]
`
`Humanization of an Anti-Vascular Endothelial Growth Factor Monoclonal
`
`Antibody for the Therapy of Solid Tumors and Other Disorders
`
`Leonard G. Presta, Helen Chen, Shane J. O’Connor, Vanessa Chisholm, Y. Gloria Meng, Lynne Krummen,
`Marjorie Winkler, and Napoleone Ferraral
`Depamnenrr of Immunology. Process Sciences, Molecular Biology. Bioanalytical Technology and Cardiovascular Research. Genenlech. Inc. South San Francisco. California
`94080
`
`ABSTRACT
`
`Vascular endothelial growth factor (VEGF) is a major mediator of
`angiogenesis associated with tumors and other pathological conditions,
`including proliferative diabetic retinopathy and age-related macular
`degeneration. The murlne anti-human VEGF monoclonal antibody
`(muMAb VEGF) A.4.6.l has been shown to potently suppress angio-
`genesis and growth in a variety of human tumor cells lines transplanted
`in nude mice and also to inhibit neovascularization in a primate model
`of ischemic retinal disease. In this report, we describe the humaniza-
`tion of muMAb VEGF A.4.6.l. by site-directed mutagenesis of a human
`framework. Not only the residues involved in the six complementarity-
`determining regions but also several framework residues were changed
`from human to murine. Humanized anti-VEGF F(ab) and IgGI vari-
`ants bind VEGF with affinity very similar to that of the original
`murine antibody. Furthermore, recombinant humanized MAb VEGF
`inhibits VEGF-induced proliferation of endothelial cells in vitro and
`tumor growth in vivo with potency and efficacy very similar to those of
`muMAb VEGF A.4.6.l. Therefore, recombinant humanized MAb
`VEGF is suitable to test the hypothesis that inhibition of VEGF-
`induced angiogenesis is a valid strategy for the treatment of solid
`tumors and other disorders in humans.
`
`INTRODUCTION
`
`it is now well established that angiogenesis is implicated in the
`pathogenesis of a variety of disorders. These include solid tumors,
`intraocular neovascular syndromes such as proliferative retinopathies
`or AMD,2 rheumatoid arthritis, and psoriasis (l, 2, 3). In the case of
`solid tumors. the neovascularization allows the tumor cells to acquire
`a growth advantage and proliferative autonomy compared to the
`normal cells. Accordingly, a correlation has been observed between
`density of microvessels in tumor sections and patient survival
`in
`breast cancer as well as in several other tumors (4—6).
`The search for positive regulators of angiogenesis has yielded
`several candidates. including acidic fibroblast growth factor (FGF),
`bFGF, transforming growth factor a, transforming growth factor B,
`hepatocyte growth factor, tumor necrosis factor-a, angiogenin. inter-
`leukin 8. and others (i. 2). However, in spite of extensive research,
`there is still uncertainty as to their role as endogenous mediators of
`angiogenesis. The negative regulators thus far identified include
`thrombospondin (7), the Mr 16,000 NHz-terminal fragment of prolac-
`tin (8), angiostatin (9), and endostatin (10).
`Work done over the last several years has established the key role
`of VEGF in the regulation of normal and abnormal angiogenesis (l l).
`The finding that the loss of even a single VEGF allele results in
`
`embryonic lethality points to an irreplaceable role played by this
`factor in the development and differentiation of the vascular system
`(1 1). Also, VEGF has been shown to be a key mediator of neovas-
`cularization associated with tumors and intraocular disorders (II).
`The VEGF mRNA is overexpressed by the majority of human tumors
`examined (12—16). In addition, the concentration of VEGF in eye
`fluids is highly correlated to the presence of active proliferation of
`blood vessels in patients with diabetic and other ischemia-related
`retinopathies (17). Furthermore, recent studies have demonstrated the
`localization of VEGF in choroidal neovascular membranes in patients
`affected by AMD (18).
`The muMAb VEGF A.4.6.l (19) has been used extensively to
`test the hypothesis that VEGF is a mediator of pathological angio-
`genesis in vivo. This high affinity MAb is able to recognize all
`VEGF isoforms (l9) and has been shown to inhibit potently and
`reproducibly the growth of a variety of human tumor cell lines in
`nude mice (1 1, 20—23). Moreover, intraocular administration of
`muMAb VEGF A.4.6.l resulted in virtually complete inhibition of
`iris neovascularization secondary to retinal ischemia in a primate
`model (24).
`A major limitation in the use of murine antibodies in human therapy
`is the anti-globulin response (25, 26). Even chimeric molecules, where
`the variable (V) domains of rodent antibodies are fused to human
`constant (C) regions, are still capable of eliciting a significant immune
`response (27). A powerful approach to overcome these limitations in
`the clinical use of monoclonal antibodies is “humanization" of the
`
`murine antibody. This approach was pioneered by Jones er al. (28)
`and Riechman et al. (29), who first transplanted the CDRs of a murine
`antibody into human V domains antibody.
`In the present article, we report on the humanization of muMAb
`VEGF A.4.6.l. Our strategy was to transfer the six CDRs, as defined
`by Kabat er a1. (30), from muMAb VEGF A.4.6.l
`to a consensus
`human framework used in previous humanizations (3l—33). Seven
`framework residues in the humanized variable heavy (VH) domain
`and one framework residue in the humanized variable light (VL)
`domain were changed from human to murine to achieve binding
`equivalent to muMAb VEGF A.4.6.l. This humanized MAb is suit-
`able for clinical trials to test the hypothesis that inhibition of VEGF
`action is an effective strategy for the treatment of cancer and other
`disorders in humans.
`
`MATERIALS AND METHODS
`
`Received 5/27/97: accepted 8/l6/97.
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advertisement in accordance with
`IS U.S.C. Section 1734 solely to indicate this fact.
`'To whom requests for reprints should be addressed. at Department of Cardio-
`vascular Research. Genentech.
`lnc.. 460 Point San Bruno Boulevard. South San
`Francisco. CA 94080. Phone: (4l5)225-2968: Fax: (4l5)225—6327; E-mail: Ferrara.
`Napoleone@gene.com.
`2 fire abbreviations used are: AMD. age-related macular degeneration: bFGF. basic
`fibroblast growth factor: VEGF. vascular endothelial growth factor; MAb. monoclonal
`antibody; muMAb. murine MAb: rhuMAb. recombinant humanized MAb; CDR. comple-
`mentarity-determining region.
`
`Cloning of Murine Mab A.4.6.l and Construction of Mouse-Human
`Chimeric Fab. Total RNA was isolated from hybridoma cells producing
`the anti-VEGF MAb A.4.6.l using RNAsol (Tel-Test) and reverse-tran-
`scribed to cDNA using Oligo-dT primer and the SuperScript ll system (Life
`Technologies. Inc., Gaithersburg. MD). Degenerate oligonucleotide primer
`pools. based of the NHz-terminal amino acid sequences of the light and
`heavy chains of the antibody. were synthesized and used as forward
`primers. Reverse primers were based on framework 4 sequences obtained
`from murine light chain subgroup KV and heavy chain subgroup ll (30).
`After PCR amplification. DNA fragments were ligated to a TA cloning
`vector (lnvitrogen. San Diego, CA). Eight clones each of the light and
`4593
`Pfizer v. Genentech
`
`Pfizer v. Genentech
`IPR2017-01488
`IPR2017—01488
`Genentech Exhibit 2021
`Genentech Exhibit 2021
`
`

`

`HUMANIZATION OF AN AN'I'LVEGF MONOCLONAL ANTIBODY
`
`heavy chains were sequenced. One clone with a consensus sequence for the
`light chain VL domain and one with a consensus sequence for the heavy
`chain VH domain were subcloned. respectively, into the pEMXl vector
`containing the human CL and CH1 domains (31). thus generating a mouse-
`human chimeric F(ab). This chimeric F(ab) consisted of the entire murine
`A.4.6.l VH domain fused to a human CH1 domain at amino acid SerHl 13,
`and the entire murine A.4.6.l VL domain fused to a human CL domain at
`
`amino acid LysL107. Expression and purification of the chimeric F(ab)
`were identical to those of the humanized F(ab)s. The chimeric F(ab) was
`used as the standard in the binding assays.
`Computer Graphies Models of Murine and Humanized F(ab)s. Se-
`quences of the VL and VH domains (Fig. l) were used to construct a computer
`graphics model of the murine A.4.6.1 VL—VH domains. This model was used
`to detemrine which framework residues should be incorporated into the hu-
`manized antibody. A model of the humanized F(ab) was also constructed to
`verify conect selection of murine framework residues. Construction of models
`was performed as described previously (32, 33).
`Construction _of Humanized F(ab)s. The plasmid pEMXl used for mu-
`tagenesis and expression of F(ab)s in Escherichia cali has been described
`previously (31). Briefly, the plasmid contains a DNA fragment encoding a
`consensus human K subgroup I light chain (VLKI-CL) and a consensus human
`subgroup III heavy chain (VHIIl-CHl) and an alkaline phosphatase promoter.
`The use of the consensus sequences for VL and VH has been described
`previously (32).
`the first F(ab) variant of humanized A.4.6.1, F(ab)-l.
`To construct
`site—directed mutagenesis (34) was performed on a deoxyuridine-containing
`template of pEMXl. The six CDRs were changed to the murine A.4.6.1
`sequence; the residues included in each CDR were from the sequence-based
`CDR definitions (30). F(ab)-1, therefore, consisted of a complete human
`framework (VL K subgroup l and VH subgroup III) with the six complete
`murine CDR sequences. Plasmids for all other F(ab) variants were con-
`structed from the plasmid template of F(ab)-1. Plasmids were transformed
`into E. coli strain XL-l Blue (Stratagene, San Diego. CA) for preparation
`of double— and single-stranded DNA. For each variant, DNA coding for
`light and heavy chains was completely sequenced using the dideoxynucle-
`otide method (Sequenase; U.S. Biochemical Corp.. Cleveland, OH). Plas-
`mids were transformed into E. coli strain 16C9, a derivative of MM294,
`plated onto Luria broth plates containing 50 ug/ml carbenicillin, and a
`single colony selected for protein expression. The single colony was grown
`in 5 ml of Luria broth—100 [ig/ml carbenicillin for 5—8 h at 37°C. The 5-m1
`culture was added to 500 ml of AP5—50 [Lg/ml carbenicillin and allowed to
`grow for 20 h in a 4-liter baffled shake flask at 30°C. AP5 media consists
`of 1.5 g of glucose. 11.0 g of Hycase SF. 0.6 g of yeast extract (certified),
`0.19 g of MgSO4 (anhydrous), 1.07 g of NH‘CI. 3.73 g of KC], 1.2 g of
`NaCl, 120 ml of l M triethanolamine. pH 7.4, to 1 liter of water and then
`sterile filtered through a 0.1-mm Sealkeen filter. Cells were harvested by
`
`centrifugation in a l-liter centrifuge bottle at 3000 X g. and the supernatant
`was removed. After freezing for l h, the pellet was resuspended in 25 ml
`of cold 10 mM Tris, 1 mM EDTA, and 20% sucrose, pH 8.0. Two hundred
`fifty ml of 0.1 M benzamidine (Sigma Chemical Co., St. Louis, MO) was
`added to inhibit proteolysis. After gentle stirring on ice for 3 h. the sample
`was centrifuged at 40,000 X g for 15 min. The supernatant was then applied
`to a protein G-Sepharose CL-4B (Pharmacia Biotech, Inc., Uppsala, Swe-
`den) column (0.5-ml bed volume) equilibrated with 10 mM Tris-l mM
`EDTA, pH 7.5. The column was washed with 10 ml of 10 mM Tris-l mM
`EDTA, pH 7.5, and eluted with 3 ml of 0.3 M glycine, pH 3.0. into 1.25 ml
`of l M Tris, pH 8.0. The F(ab) was then buffer exchanged into PBS using
`a Centricon-30 (Amicon, Beverly. MA) and concentrated to a final volume
`of 0.5 ml. SDS-PAGE gels of all F(ab)s were run to ascertain purity, and
`the molecular weight of each variant was verified by electrospray mass
`spectrometry.
`Construction, Expression, and Purification of Chimeric and Human-
`ized lgG Variants. For the generation of human IgGl variants of chimeric
`(chIgGl) and humanized (rhuMAb VEGF) A.4.6.l, the appropriate murine or
`humanized VL and VH (F(ab)-12; Table 1) domains were subcloned into
`separate, previously described pRK vectors (35). The DNA coding for the
`entire light and the entire heavy chain of each variant was verified by
`dideoxynucleotide sequencing.
`For transient expression of variants. heavy and light chain plasmids were
`cotransfected into human 293 cells (36) using a high efficiency procedure (37).
`Media were changed to serum free and harvested daily for up to 5 days.
`Antibodies were purified from the pooled supematants using protein A-
`Sepharose CL-4B (Pharrnacia). The eluted antibody was buffer exchanged into
`PBS using a Centricon-30 (Amicon). concentrated to 0.5 ml. sterile filtered
`using a Millex-GV (Millipore, Bedford, MA), and stored at 4°C.
`For stable expression of the final humanized IgGl variant (rhuMAb
`VEGF), Chinese hamster ovary (CHO) cells were transfected with dicis-
`tronic vectors designed to coexpress both heavy and light chains (38).
`Plasmids were introduced into DP12 cells, a proprietary derivative of the
`CHO-Kl DUX 811 cell line developed by L. Chasin (Columbia University,
`New York. NY), via lipofection and selected for growth in glycine/
`hypoxanthine/thymidine (GHT)-free medium (39). Approximately 20 un-
`amplified clones were randomly chosen and reseeded into 96-well plates.
`Relative specific productivity of each colony was monitored using an
`ELISA to quantitate the full-length human lgG accumulated in each well
`after 3 days and a fluorescent dye, Calcien AM, as a surrogate marker of
`viable cell number per well. Based on these data, several unamplified
`clones were chosen for further amplification in the presence of increasing
`concentrations of methotrexate. Individual clones surviving at 10. 50, and
`100 nM methotrexate were chosen and transferred to 96-well plates for
`productivity screening. One clone. which reproducibly exhibited high spe—
`cific productivity, was expanded in T-flasks and used to inoculate a spinner
`
`Table 1 Binding of humanized anti-VEGF F(ab) variants to VEGF“
`
`Variant
`chim-F(ab)
`F(ab)-l
`F(ab)-2
`
`F(abH
`
`F(ab)-4
`
`F(abys
`F(ab)«6
`F(ab)-7
`F(ab)-8
`
`Template
`Chimeric F(ab)
`Human FR
`
`Changesb
`
`F(ab)-l
`
`F(ab)-4
`F(ab)«5
`F(ab)-5
`F(ab)—5
`
`ArgH71_Lg
`AspH73fl
`hut/WE
`LeuH78Ala
`namfi
`[leH69E
`LeuH78Ala
`
`Purpose
`1.0
`Straight CDR swap
`Chimera light chain
`F(ab)-1 heavy chain
`F(ab)-1 light chain
`Chimera heavy chain
`CDR-H2 conformation
`Framework
`VL-VH interface
`CDR-H1 conformation
`CDR-HZ conformation
`CDR-H2 conformation
`CDR-H1 conformation
`
`Mean
`
`>1350
`>145
`
`2.6
`
`>295
`
`80.9
`36.4
`45.2
`9.6
`
`ECSO F(ab)-X
`
`ECSO chimeric F(ab)‘
`SD
`
`0.1
`
`6.5
`4.2
`2.3
`0.9
`
`>150
`CDR—H2 conformation
`flfl49fl
`F(ab)-8
`F(ab)-9
`6.4
`Framework
`Aan76Ser
`F(ab)-8
`F(ab)- 10
`3.3
`Framework
`LysH75ATa
`F(ab)-10
`F(ab)-l 1
`1.6
`CDR-H3 conformation
`ArgH94L—ys
`F(ab)-10
`F(ab)-12
`" Anti-VEGF F(ab) variants were incubated with biotinylated VEGF and then transferred to ELISA plates coated with KDR-lgG (40).
`b Murine residues are underlined; residue numbers are according to Kabat et a1. (30).
`‘ Mean and SD are the average of the ratios calculated for each of the independent assays; the EC50 for chimeric F(ab) was 0.049 1‘ 0.013 mg/ml (1.0 M).
`4594
`
`l .2
`0.4
`0.6
`
`N
`
`2
`3
`
`2
`
`3
`
`2
`2
`2
`4
`
`2
`4
`2
`4
`
`

`

`HUMANIZATION OF AN ANTI~VEGF MONOCLONAL ANTIBODY
`
`antibiotics (growth medium). essentially as described previously (42). For
`mitogenic assays, endothelial cells were seeded at a density of 6 X I03
`cells/well in 6—well plates in growth medium. Either muMAb VEGF A.4.6.l or
`rhuMAb VEGF was then added at concentrations ranging between I and 5000
`ng/ml. After 2—3 h. purified E. cali-expressed rhVEGF”,5 was added to a final
`concentration of 3 ng/ml. For specificity control, each antibody was added to
`endothelial cells at the concentration of 5000 ng/ml, either alone or in the
`presence of 2 ng/ml bFGF. After 5 or 6 days. cells were dissociated by
`exposure to trypsin. and duplicate wells were counted in a Coulter counter
`(Coulter Electronics. Hialeah. FL). The variation from the mean did not exceed
`10%. Data were analyzed by a four-parameter curve fitting program (Kalei-
`daGraph).
`In Vivo Tumor Studies. Human A673 rhabdomyosarcoma cells (Amer-
`ican Type Culture Collection; CRL l598) were cultured as described
`previously in DMEM/FIZ supplemented with 10% fetal bovine serum. 2
`mM glutamine. and antibiotics (20, 22). Female BALB/c nude mice. 6-40
`weeks old. were injected s.c. with 2 X 10" tumor cells in the dorsal area in
`a volume of 200 [1.1. Animals were then treated with muMAb VEGF
`A.4.6.l. rhuMAb VEGF. or a control murine MAb directed against the
`gpl20 protein. Both anti-VEGF MAbs were administered at the doses of
`0.5 and 5 mg/kg; the control MAb was given at the dose of 5 mg/kg. Each
`MAb was administered twice weekly i.p. in a volume of mo pl. starting
`24 h after tumor cell inoculation. Each group consisted of to mice. Tumor
`size was determined at weekly intervals. Four weeks after tumor cell
`inoculation. animals were euthanized. and the tumors were removed and
`weighed. Statistical analysis was performed by ANOVA.
`
`RESULTS
`
`culture. After several passages. the suspension-adapted cells were used to
`inoculate production cultures in GHT-containing. serum-free media sup-
`plemented with various hormones and protein hydrolysates. Harvested cell
`culture fluid containing rhuMAb VEGF was purified using protein A-
`Sepharose CL-4B. The purity after this step was ~99%. Subsequent
`purification to homogeneity was carried out using an ion exchange chro-
`matography step. The endotoxin content of the final purified antibody was
`<0.IO eu/mg.
`F(ab) and IgG Quantitation. For quantitating F(ab) molecules. ELISA
`plates were coated with 2 [Lg/ml of goat anti-human lgG Fab (Organon
`Teknika. Durham. NC) in 50 mM carbonate buffer. pH 9.6. at 4°C overnight
`and blocked with PBS-0.5% BSA (blocking buffer) at room temperature for
`I h. Standards [0.78—50 ng/ml human F(ab)] were purchased from Chemicon
`(Temecula. CA). Serial dilutions of samples in PBS-0.5% BSA-0.05% poly-
`sorbate 20 (assay buffer) were incubated on the plates for 2 h. Bound F(ab) was
`detected using horseradish peroxidase-labeled goat anti-human lgG F(ab)
`(Organon Teknika), followed by 3.3’,5.5'-tetramethylbenzidine (Kirkegaard &
`Perry Laboratories. Gaithersburg. MD) as the substrate. Plates were washed
`between steps. Absorbance was read at 450 nm on a VM plate reader
`(Molecular Devices. Menlo Park. CA). The standard curve was fit using a
`four—parameter nonlinear
`regression curve-fitting program developed at
`Genentech. Data points that fell in the range of the standard curve were used
`for calculating the F(ab) concentrations of samples.
`The concentration of full-length antibody was determined using goat anti-
`human IgG Fe (Cappel. Westchester. PA) for capture and horseradish perox-
`idase-labeled goat anti-human Fc (Cappel) for detection. Human IgGl (Chemi-
`con) was used as standard.
`VEGF Binding Assays. For measuring the VEGF binding activity of
`F(ab)s, ELISA plates were coated with 2 ug/ml rabbit F(ab’)2 to human
`IgG Fc (Jackson ImmunoResearch. West Grove. PA) and blocked with
`blocking buffer (described above). Diluted conditioned medium containing
`3 ng/ml of KDR-IgG (40) in blocking buffer were incubated on the plate for
`I h. Standards [6.9—440 ng/ml chimeric F(ab)] and 2-fold serial dilutions
`of samples were incubated with 2 nM biotinylated VEGF for l h in tubes.
`The solutions from the tubes were then transferred to the ELISA plates and
`incubated for l h. After washing. biotinylated VEGF bound to KDR was
`detected using horseradish peroxidase-labeled streptavidin (Zymed. South
`San Francisco. CA or Sigma) followed by 3.3'.5.5'-tetramethylbenzidine as
`the substrate. Titration curves were fit with a four-parameter nonlinear
`regression curve-fitting program (KaleidaGraph; Synergy Software. Read—
`ing. PA). Concentrations of F(ab) variants corresponding to the midpoint
`absorbance of the titration curve of the standard were calculated and then
`
`divided by the concentration of the standard corresponding to the midpoint
`absorbance of the standard titration curve. Assays for full-length IgG were
`the same as for the F(ab)s except that the assay buffer contained l0%
`human serum.
`
`Humanization. The consensus sequence for the human heavy
`chain subgroup III and the light chain subgroup K I were used as the
`framework for the humanization (Ref. 30; Fig. I). This framework has
`been successfully used in the humanization of other murine antibodies
`(3l. 32. 43, 44). All humanized variants were initially made and
`screened for binding as F(ab)s expressed in E. coli. Typical yields
`from 500-ml shake flasks were 0.1—0.4 mg F(ab).
`Two definitions of CDR residues have been proposed. One is based
`on sequence hypervariability (30) and the other on crysz structures
`of F(ab)-antigen complexes (45). The sequence-based CDRs are
`larger than the structure-based CDRs, and the two definitions are in
`agreement except for CDR-HI; CDR—H1 includes residues H3I-H35
`according to the sequence-based definition. and residues H26—H32
`according to the structure-based definition (light chain residue num-
`bers are prefixed with L; heavy chain residue numbers are prefixed
`BlAcore Biosensor Assays. VEGF binding of the humanized and chimeric
`with H). We. therefore, defined CDR-HI as a combination of the two,
`F(ab)s were compared using a BlAcore biosensor (4l). Concentrations of
`i.e.. including residues H26 —H35. The other CDRs were defined using
`F(ab)s were determined by quantitative amino acid analysis. VEGF was
`the sequence-based definition (30).
`coupled to a CM-5 biosensor chip through primary amine groups according to
`The chimeric F(ab) was used as the standard in the binding assays.
`manufacturer‘s instructions (Pharmacia). Off-rate kinetics were measured by
`In the initial variant. F(ab)-l, the CDR residues were transferred from
`saturating the chip with F(ab) [35 p.l of 2 uM F(ab) at a flow rate of 20 ul/min]
`the murine antibody to the human framework and, based on the
`and then switching to buffer (PBS—0.05% polysorbate 20). Data points from
`models of the murine and humanized F(ab)s. the residue at position
`0—4500 5 were used for off-rate kinetic analysis. The dissociation rate constant
`H49 (Ala in humans) was changed to the murine Gly. In addition,
`(km) was obtained from the slope of the plot of ln(R0/R) versus time, where R0
`F(ab)s that consisted of the chimeric heavy chain/F(ab)-l light chain
`is the signal at t = 0 and R is the signal at each time point.
`On-rate kinetics were measured using 2-fold serial dilutions of F(ab)
`[F(ab)-2] and F(ab)-l heavy chain/chimeric light chain [F(ab)-3] were
`(0.0625—2 mM). The slope. K,. was obtained from the plot of ln(-dR/dt)
`generated and tested for binding. F(ab)-I exhibited a binding affinity
`versus time for each F(ab) concentration using the BlAcore kinetics eval—
`greater than lOOO-fold reduced from the chimeric F(ab) (Table 1).
`uation software as described in the Pharmacia Biosensor manual. R is the
`Comparing the binding affinities of F(ab)-2 and F(ab)—3 suggested
`signal at time I. Data between 80 and I68. I48. l28. I I4, 102. and 92 s were
`that framework residues in the F(ab)-l VH domain needed to be
`used for 0.0625. 0.125. 0.25. 0.5. l, and 2 mM F(ab). respectively. The
`altered to increase binding.
`association rate constant (km) was obtained from the slope of the plot of K5
`Previous humanizations (3|. 32, 43. 44) as well as studies of
`versus F(ab) concentration. At the end of each cycle. bound F(ab) was
`F(ab)-antigen crystal structures (45. 47) have shown that residues H7]
`removed by injecting 5 pl of 50 mM HCI at a flow rate of 20 ul/min to
`and H73 can have a profound effect on binding. possibly by influ-
`regenerate the chip.
`encing the conformations of CDR-HI and CDR-H2. Changing the
`Endothelial Cell Growth Assay. Bovine adrenal cortex-derived capillary
`endothelial cells were cultured in the presence of low glucose DMEM (Life
`human residues to their murine counterparts in F(ab)-4 improved
`Technologies. Inc.) supplemented with 10% calf serum. 2 mM glutamine. and
`binding by 4-fold (Table 1). Inspection of the models of the murine
`4595
`
`

`

`HUMANIZATION OF AN ANTI-VEGF MONOCLONAL ANTIBODY
`
`Variable Heavy
`
`A. 4 . 6 . 1
`
`P (ab) - 12
`
`humI I I
`
`E IQLVQSGPELKQPGETVRISCKASGXIETNXGMNWVKQAPGKGLKWMG
`t
`t
`* I t
`t i k i
`*
`fl *
`EVOLVESGGGLVQPGGSLRLSCAASWMNWVRQAP GKGLEWVG
`i
`t i
`t
`t
`EVOLVESGGGLVQPGGSLRLSCAASGE‘TFSS YAMSWVRQAPGKGLEWVS
`1
`1 0
`2 0
`3 0
`4 0
`
`A. 4 . 6 . 1 WTFSLETSASTAYLQISNLKNDDTATYFCAK
`* t i R t 'k
`i i
`P (ab) - 12 WTFSLDTSKSTAYLQMNSLRAEDTAVYYCAK
`* ttti it!
`it! t
`i i
`'k
`t *
`v1 SGDGGs'rvaDSVKGRF'rIsanNSKN'rLYLmiNSLRAEDTAWXCAR
`5 0
`a
`60
`7 0
`8 0
`abc
`90
`
`humI I I
`
`A - 4 - 6 - 1 WGAGTTVTVSS
`i
`*
`F (ab) -12 WGQGTLVTVSS
`*
`t
`G——————————FDYWGQGTLVTVSS
`1 10
`
`humI I I
`
`A. 4 . 6 . 1
`
`Variable Light
`DIQMTQTTSSLSASLGDRVIISCSASQQISWWYQQKPDGTVKVLIY
`* i
`t
`i *
`* i t i
`F (ab) — 12 DIQMTQSPSSLSASVGDRVT ITCWQQKPGKAPKVLIY
`t
`t
`t
`*
`
`humKI
`
`DIQMTQSPSSLSASVGDRVTITCRASQSISNYLAWYQQKPGKAPKLLIY
`1
`1 o
`20
`30
`4 o
`
`A. 4 . 6 . 1
`
`EISSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATvvcmxammF
`I t
`t t
`t
`
`* i
`t
`“1:
`F (ab) — 12 WGVPSRFSGSGSGTDFTLTISSLQPEDE‘ATYYCQQXSMEIFy
`humKI
`AASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPWTF
`5 0
`60
`7 0
`8 0
`90
`
`A.4.6.1
`
`E‘ (ab) -12
`
`GGGTKLEIKR
`t
`t
`GQGTKVEIKR
`
`humKI
`
`GQGTKVE IKR
`1 00
`
`Fig. 1. Amino acid sequence of variable heavy and light domains of muMAbVEGF
`A.4.6.l, humanized F(ab) with optimal VEGF binding [F(ab)-12] and human consensus
`frameworks (humlll. heavy subgroup [I]; humxl, light K subgroup I). Asterisks. differ-
`ences between humanized F(ab)-12 and the murine MAb or between F(ab)-12 and the
`human framework. CDRs are underlined.
`
`and humanized F(ab)s suggested that residue L46, buried at the
`VL—VH interface and interacting with CDR-H3 (Fig. 2), might also
`play a role either in determining the conformation of CDR-H3
`and/or affecting the relationship of the VL and VH domains. When
`the murine Val was exchanged for the human Leu at L46 [F(ab)-5],
`the binding affinity increased by almost 4-fold (Table 1). Three
`other buried framework residues were evaluated based on the
`
`H94, human and murine sequences most often have an Arg (30). In
`F(ab)-12, this Arg was replaced by the rare Lys found in the murine
`antibody (Fig. 1), and this resulted in binding that was less than 2-fold
`from the chimeric F(ab) (Table 1). F(ab)-12 was also compared to the
`chimeric F(ab) using the BIAcore system (Phannacia). Using this
`technique, the Kd of the humanized F(ab)-12 was 2-fold weaker than
`that of the chimeric F(ab) due to both a slower kon and faster koff
`(Table 2).
`Full-length MAbs were constructed by fusing the VL and VH
`domains of the chimeric F(ab) and variant F(ab)-12 to the constant
`domains of human K light chain and human IgG1 heavy chain. The
`full-length 12-IgGl [F(ab)—12 fused to human IgGl] exhibited bind-
`ing that was 1.7-fold weaker than the chimeric IgGl (Table 3). Both
`lZ-IgGl and the chimeric lgGl bound slightly less well than the
`original muMAb VEGF A.4.6.1 (Table 3).
`Biological Studios. rhuMAb VEGF and muMAb VEGF A.4.6.l
`were compared for their ability to inhibit bovine capillary endo-
`thelial cell proliferation in response to a near maximally effective
`concentration of VEGF,“ (3 ng/ml). In several experiments, the
`two MAbs were found to be essentially equivalent, both in potency
`and efficacy. The EDsos were, respectively, 50 t 5 and 48 t 8
`ng/ml (~0.3 nM). In both cases, 90% inhibition was achieved at the
`concentration of 500 ng/ml (~3 nM). Fig. 3 illustrates a represent-
`ative experiment. Neither muMAb VEGF A.4.6.1 nor rhuMAb
`VEGF had any effect on basal or bFGF-stimulated proliferation of
`capillary endothelial cells (data not shown), confirming that the
`inhibition is specific for VEGF.
`To determine whether similar findings could be obtained also in an
`in vivo system. we compared the two antibodies for their ability to
`suppress the growth of human A673 rhabdomyosarcoma cells in nude
`mice. Previous studies have shown that muMAb VEGF A.4.6.1 has a
`
`dramatic inhibitory effect in this tumor model (20, 22). As shown in
`Fig. 4, at both doses tested (0.5 and 5 mg/kg), the two antibodies
`markedly suppressed tumor growth as assessed by tumor weight
`measurements 4 weeks after cell inoculation. The decreases in tumor
`
`weight compared to the control group were, respectively, 85 and 93%
`at each dose in the animals treated with muMAb VEGF A.4.6.l versus
`90 and 95% in those treated with rhuMAb VEGF. Similar results were
`obtained with the breast carcinoma cell line MDA-MB 435 (data not
`shown).
`
`DISCUSSION
`
`The murine MAb A.4.6.1, directed against human VEGF (42),
`was humanized using the same consensus frameworks for the light
`and heavy chains used in previous humanizations (31, 32, 43, 44),
`i.e., VKI and VHIII (30). Simply transferring the CDRs from the
`murine antibody to the human framework resulted in a F(ab) that
`exhibited binding to VEGF reduced by over lOOO-fold compared to
`the parent murine antibody. Seven non-CDR, framework residues
`in the VH domain and one in the VL domain were altered from
`
`molecular models: H49, H69, and H78. Position H69 may affect
`the conformation of CDR-H2, whereas position H78 may affect the
`conformation of CDR-H1 (Fig. 2). When each was individually
`changed from the human to murine counterpart, the binding im-
`human to murine to achieve binding equivalent
`proved by 2-fold in each case [F(ab)-6 and F(ab)-7; Table 1]. When
`murine antibody.
`both were simultaneously changed, the improvement in binding
`In the VH domain, residues at positions H49, H69, H71, and H78
`was 8—fold [F(ab)-8; Table 1]. Residue H49 was originally in-
`are buried or partially buried and probably effect binding by
`cluded as the murine Gly; when changed to the human consensus
`influencing the conformation of the CDR loops. Residues H73 and
`counterpart Ala,
`the binding was reduced by 15-fold [F(ab)-9;
`Table 1].
`H76 should be solvent exposed (Fig. 2) and hence may interact
`directly with the VEGF; these two residues are in a non-CDR loop
`We have found during previous humanizations that residues in a
`adjacent to CDRs H1 and H2 and have been shown to play a role
`framework loop, FR-3 (30) adjacent to CDR-H1 and CDR-H2, can
`in binding in previous humanizations (31, 32, 44). The requirement
`affect binding (44). In F(ab)-10 and F(ab)-l 1, two residues in this loop
`were changed to their murine counterparts: Aan76 to murine Ser
`for lysine at position H94 was surprising given that this residue is
`[F(ab)-10] and LysH75 to murine Ala [F(ab)-ll]. Both effected a
`arginine in the human framework (Fig. 1). In some crystal struc-
`relatively small improvement in binding (Table 1). Finally, at position
`tures of F(ab)s, ArgH94 forms a hydrogen-bonded salt-bridge with
`4596
`
`to the parent
`
`

`

`HUMANIZATION OF AN ANTI-VEGF MONOCLONAL ANTIBODY
`
`
`
`Fig. 2. Ribbon diagram of the model of humanized F(ab)-12 VL and VH domains. VL domain is shown in brown with CDRs in tan. The side chain of residue [/46 is shown in
`yellow. VH domain is shown in purple with CDRs in pink. Side chains of VH residues changed from human to murine are shown in yellow.
`
`Table 2 Binding ofanli-VEGF F(ab) variants to VEGF using the BlAcare system"
`Amount of (Fab)
`Kd (m)
`km (M_'s_')
`1:0,Y (s-l)
`bound (RU)
`Variant
`0.91
`6.5 x 10“
`5.9 x 10'5
`4250
`chain-F(ab)"
`l.8
`3.5 x 10“
`6.3 x to"
`3740
`F(ab)—12
`" The amount of F(ab) bound. in resonance units (RU). was measured using a BlAcore
`system when 2 pg F(ab) was injected onto a chip containing 2480 RU of immobilized
`VEGF. Off-rate kinetics (km) were measured by saturating the chip with F(ab) and then
`monitoring dissociation after switching to buffer. On-rate kinetics (Iron) were measured
`using 2-fold serial dilutions of F(ab). Kd. the equilibrium dissociation constant. was
`calculated as knnlkm.
`b chim-F(ab) is a chimeric F(ab) with murine VL and VH domains fused to human CL
`and CHI heavy domains.
`
`AsleOl (33. 48). Substitution of lysine for arginine might con—
`ceivably alter this salt-bridge and perturb the conformation of
`CDR-H3.
`
`In the VL domain, only one framework residue had to be changed
`to murine to optimize the humanization. Position L46 is at the VL-Vl-l
`interface. where it is buried and interacts directly with CDR-H3 (Fig.
`2). The requirement for murine valine (as opposed to human leucine)
`implies that this residue plays an important role in the conformation of
`CDR-H3. The necessity of retaining LysH94 in VH, which is also
`4597
`
`adjacent to CDR-H3, suggests that CDR-H3 plays a major role in the
`binding of the antibody to VEGF.
`The humanized version with optimal binding, lZ-lgGl, exhib-
`ited only a 2-fold reduction in binding compared to the parent
`murine antibody (Table 3). An analysis of the binding kinetics of
`the humanized and chimeric F(ab)s showed that both had similar
`off-rates but that the humanized F(ab) had a 2-fold slower on-rate
`(Table