1
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`EX2100
`Eli Lilly & Co. v. Teva Pharms. Int'l GMBH
`IPR2018-01423
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`942
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`C. CHIC/[IS er (IL/Molecular Immunology 39 (2003) 94 14952
`
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`procedure. Mild losses are normally associated with this
`procedure (Carter et al., 1992; Hsiao et al., 1994). A lower
`affinity is normally tolerated for humanized antibodies.
`These effects have been partially remedied by reengineer—
`ing the framework (Kettleborough et al., 1991; Singer et al.,
`1993; Nakatani et al., 1994; Zhu and Carter, 1995; Saldanha
`et al., 1999). Therefore, it is crucial to describe more sub-
`tle structural features that allow the preservation of antibody
`paratope integrity during thisprocess. The rules are far from
`being completely understood. Long—range effects promoted
`by certain residues are among those forces responsible for
`subtle changes in the global structure. Unfortunately these
`are the most difficult issues to probe, since even small devia—
`tions could be responsible for removing residue—residue con—
`tact, or reducing surface complementarity. Therefore, even
`distant residues could interfere with paratope conformation
`and antigen binding (Chien et al., 1989).
`We used an anti-human CD18 as a model antibody for
`humanization. CD18 is an integrin family membrane protein
`involved in cell adhesion. It is found in many different cell
`types and it is always associated with one of the different
`isoforms of CD11. Antibodies to CD18 may inhibit cell—cell
`attachment and especially leukocyte-tissue adhesion. There—
`fore, anti—CD18 antibodies have been proposed as adjuvant
`in many therapies that involves leukocytes infiltration and
`inflammation. They have been tested successfully as protec-
`tive agents in ischemic myocardial injury in animal models
`(Gao et al., 2002), but much of this enthusiasm was lost
`after failure of human trials (Dove, 2000). Its potential ap—
`plication is much wider, and it has also been cited as a po—
`tential treatment for preventing meningitis sequels or graft
`rejection (Tuomanen et al., 1989; lsobe et al., 1997). The
`antibody used in this work, mAb 6.7, binds to CD18 (David
`et al., 1991) in a unique inhibitory epitope recently mapped
`to residues 350—432 (Lu et al., 2001).
`_
`In this work we describe the complete humanization of
`murine mAb 6.7 anti—human CD18. In a previous paper we
`described the successful humanization of the VH domain
`
`by means of a germline human VH gene fragment sequence
`using an expanded CDRl (CDRl +H1) graft (Caldas et al.,
`2000). We apply this same concept to the design of two ver-
`sions of the humanized VL domain. While testing them, we
`have shown the effect of a mutation far from the antibody’s
`
`binding site that resulted in a loss of affinity for the intact
`CD18 molecule on the cell surface. We propose it could be
`the result of a new, unexpected effect of a residue that inter—
`feres with binding of the completely humanized scFV even
`though it is located at a distance from the binding site.
`
`2. Materials and methods
`
`2.]. Computational analysis
`
`Similarity analysis was initially perfoi‘med'using FASTA
`(Pearson, 2000) and the Swiss-,protdatabaSe (Bairoch and
`
`
`preiler, 2000). Blastp (Altschul et al., 1997) was also used
`either through the IgBlast page (http://www.ncbi.nlm.nih,
`gov/igblast/) or to analyze the PDB database (Berman et al.,
`
`2000). Amino acid residues usage in mouse and human VL
`was calculated from all-mouse and all-human VL files from
`
`Kabat Database (Johnson and Wu, 2000), using perl scripts,
`
`Germline VL sequences were obtained from the IgBlast
`page. Clustal W (Higgins et al., 1996) was used for multi-
`
`sequence alignment that was Visualized using BioEdit ver.
`
`sion 5.0.9 (http://www.mbio.ncsu.edu/bioedit/bioedit.html)
`Tri-dimensional structure was visualized using RASMOL
`version 2.6 (Bernstein, 2000). This version of RASMQL
`permits direct distance calculations, but we also used per
`scripts to perform such calculations. Accessibility of each
`atom in PDB file was calculated using the program Surfrace
`version 1.1 (Tsodikov et al., 2002). Variable region number
`ing follows Kabat’s convention (Kabat et al., 1991).
`
`
`
`
`
`
`2.2. Synthetic oligonucleotides
`
`The overlapping oligonucleotides used for the synthe
`sis of the humanized versions were supplied by DNA
`
`
`
`gency (Malvern, PA). The oligonucleotides used were: L1
`(5’ AGAAGATCTGACGTGGTTATGACCCAAAGCCCC-
`TTGTCCCTGCCAGTCACTCTGGGC3’); L2 (5’GTGCA
`
`CCAAGCGTTGGCTA GACCTGCAGCTTATAGAGGCA
`GGCTGGCCCAGAGTGACTGG3’); L3L (5’CAACGCTT
`GGTGCACACCAACGGTAACACCTACTTCCACTGGT—
`
`TTCTTCAAAGACCAGGACAG3’); L3Q (5’ CAACGCTT
`GGTGCACACCAACGGTAACACCTACTTCCACTGGT—
`
`
`
`
`TTCAACAAAGACCAGGACAG3’); L4 (5/AAAGAATCT
`ATTGGAAACCTTGTAAATCAACAGACGGGGGCTCT4
`
`GTCCTGGTCTTTGS’); L5 (5’ TCCAATAGATTCTTTGG‘
`
`AGTCCCAGACAGGTTTTCTGGCTCTGGTAGCGGGA—
`
`
`
`
`~ CTGATTTC3’); L6 (5/ATACACCCCGACATCCTCAGCT
`TCTACCCTGGAAATTTTGAGTGTGAAATCAGTCCC-
`
`GCT3’); L7 (5/GATGTCGGGGTGTATTATTGTTCACAG
`TCAACACATGTTCCCCGGACTTTCGGTGGTGGC3’ );
`L8 (5’ACCATGGGCTCTCTTGATCTCGAGCTTTGTGC
`CACCACCGAAAGT3’); EXTLl (S/GC TAGTAGAAGAT
`CT3’) and EXTL2 (5’CACACCATGGGCTCT3’).
`The 5’ L1 oligonucleotide contains a BglII restriction sit
`
`and the 3’ L8 carries a Ncolpand a Xhol site (these sites are_,_:______\
`underlined). The L3L and L3Q contain the codon change be-
`L
`tween the two VL humanized versions in bold (see results).
`
`
`
`
`_,
`
`2.3. Assembly of the VL humanized versions»
`
`The 6.7 VL sequence was previously determined (Caldas
`et al., 2000) and its amino acid sequence was used for a :-
`search for the closest human germline sequence. The closest
`human germline sequence chosen was used as a framework
`to graft the murine CDRs. The DNA fragments for tWO
`humanized VL versions were generated using eight overlap-
`ping oligonucleotides ranging from 45 to 63 bp, with 15 bp
`of complementarity, in a PCR~based—pr0tocol. Aliquots of .1
`
`_
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`C. Caldas et al. /Molecular Immunology 59 (2003) 941~~952
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`943
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`10pmol of each pair of complementary oligonucleotides
`were annealed separately in a 50 ul reaction containing
`9111M Tris—HCl (pH 7.6), 13 mM MgC12, 21 mM DTT and
`200 ptM dNTPs. The samples were incubated in 400 ml of _
`‘ boiling water for 5min and left standing ,until the water
`reached room temperature. Each pair of'primers were elon-
`' gated by the addition of 24U DNA polymerase I (Klenow
`fragment, Biolabs) for 30 min at room temperature. The two
`airs of primers coding for the N-terminus were mixed and
`7 amplified by PCR, and the same procedure was followed
`for the two pairs of primers coding for the C—terminus. The
`DNA fragments for the two N—terminus and C—terminus re-
`- gions were amplified by 20 thermal cycles of 94 0C for 30 s,
`60 0C for 405 and 72 0C for 2min and analyzed in agarose
`gel. Finally, the full-length DNA fragment was amplified us-
`ing as DNA templates the amplified fragments from the first
`PCR, extracted directly from the agarose gel with a pipet
`tip. The 5’ and 3’ external primers (EXTLI and EXTLZ)
`were used in this second PCR; the DNA fragments were
`PCR—amplified after 25 thermal cycles of 94°C for 30 s,
`60 0C for 40 s and 72 0C for 2 min, purified with Quiaquick
`gel purification system (QIAGEN), using the manufacturer’s
`recommendation, cloned in the pGEM-T vector (Promega)
`and sequenced using the T7 Sequencing Kit (Pharmacia).
`
`2.4. Construction of the expression vectors
`
`The constructs that code for the humanized scFvs were as—
`
`sembled based on the plg17hVH/mVL (Caldas et al., 2000)
`derived from pIg17Z22 (Brigido et al., 1993). In this sys-
`tem, the proteins can be expressed as a fusion product with
`a staphylococcal protein A domain, in order to allow the de—
`tection of the recombinant protein and also to facilitate the
`purification in an IgG sepharose chromatography column.
`The DNA fragments of the humanized VL were cloned in the
`vector p1g17hVH/mVL, which had its murine VL replaced
`by the humanized VLs, through digestion with Bng and
`Ncol. After the construction and verification of the expres-
`sion cassettes, these were transferred to the Pichia pastoris
`expression vector pPIg16 (Andrade et al., 2000) by replac-
`ing the existing scFv, through digestion with the restriction
`enzymes Xmal and EcoRI.
`
`2.5. Expression of the humanized scFvs in Pichia pastoris
`
`L
`
`_
`‘-
`
`
`
`
`P. pastoris G81 15 cells (Invitrogen, San Diego, CA) were
`grown in liquid medium and made competentby resuspen-
`sion in 1M sorbitol. The cells were eletroporated by pulse
`discharge (1500V, 25 ME 400 S2; Bio-Rad Gene Pulser) for
`5 ms in the presence of 5—10 ug of plasmidDNA linearized
`_ with SalI. This enzyme cuts withinthe plasmid—encoded
`H154 gene and favors homologous recombination with
`the endogenous, non—functional his4 geneof GSllS, cells.
`Therefore,
`transformants (His+) were screened by their
`capacity to grow in the absence of histidine‘as described
`by the manufacturer (Invitrogen).'Pro:teiri expression kinet-
`
`
`
`ics were determined by growing clones expressing the two
`humanized scFvs in 25 m1 of BMGY medium (1% yeast
`extract, 2% peptone, 10mM potassium phosphate, pH 6.0,
`1,34% yeast nitrogen base, 4 X _10_5% biotin, 1% glycerol)
`at 30 0C in a shaking incubator (250 rpm) until the culture
`reached A600 = 2.0—6.0. Cells were then centrifuged and
`resuspended in 100—200 ml of BMMY medium (which
`has 0.5% methanol instead of 1% glycerol of the BMGY
`medium, while the other components are the same) to in—
`duce protein expression. Cells were incubated for 4 days at
`30 0C in a shaking incubator (250 rpm). Aliquots of culture
`supernatants were taken daily, and examined by SDS—PAGE
`and Western blotting. For large scale expression, the clones
`were grown exactly the same way as above, for 80h at
`30 0C under agitation. The supernatants were harvested
`following centrifugation and filtration through a 0.45 um
`cellulose acetate filter. After the addition of 80 ug of Pep—
`statin A and 14 (Lg of PMSF to the supernatants, these were
`concentrated to about 5m1 using an ultrafiltrating stirred
`cell.(Corning) with a membrane filter with a .cut-off of
`10,000 Da according to manufacturer’s instructions.
`‘
`
`2.6. Purification of recombinant scFvs
`
`The concentrated supernatants were run through an IgG
`Sepharose 6B Fast Flow column (Pharmacia) previously ac—
`tivated by three alternating washes with 0.5 M acetic acid, pH
`3.4, and PBST (PBS and Tween 20, 0.1%) and finally equi-
`librated with PBS. ScFv fragments were eluted with 0.5 M
`acetic acid, immediately neutralized with 1.5M TristCl,
`pH 8.8. The purified proteins were dialyzed against PBS and
`quantified using the BCA Protein Assay Kit (Pierce).
`
`2.7. Flow cytometric analysis
`
`Peripheral blood mononuclear cells (PBMC) obtained
`from a normal individual by gradient centrifugation were
`used for immunofluorescence assays. Antibodies utilized
`were: recombinant Z22 scFv (Andrade et al., 2000) as a
`negative control; rabbit anti-human IgG-FITC (Dakopatts,
`Denmark;used in the second step of the indirect immunoflu—
`orescence reaction to bind to the protein A domain present
`in the recombinant anti—CD18 scFvs, through the Fc frag-
`ment); rabbit anti-mouse IgG (Sigma); 6.7 anti—CD18 FITC
`(Instituto Butantan—InCor, Brazil); anti—CD19PE (Dakopatts,
`Denmark); anti—CD3 FITC (Dakopatts, Denmark); anti-CD4
`FITC (Dakopatts, Denmark); anti-CD8 FITC (Dakopatts,
`Denmark) and anti—CD45RO PE (Pharmingen). The sample
`incubated with both anti-CD19PE and rabbit anti-human
`
`IgG—FITC was used to evaluate binding of the rabbit an—
`tibody to IgG expressed on B cells and exclude any other
`unspecific binding from the tests with the scFvs. Anti-CD3
`was used as a positive control of the assays. 2 X 105 cells
`were incubated with the different antibodies for 30min
`_ at 40C and washed three times. For the samples with the
`recombinant humanized scFvs, a second incubation was.
`
`3
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`

`

`944
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`C. Cu/das er (IL/Molecular lmnumology 39 (2003) 941—952
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`
`
`et al., 2000). We used the FASTA program to search [’01
`human VK sequences deposited in the Swiss-prot databage
`
`The closest human sequence found was the germline VK
`fragment KV2F (Klobeck et al., 1985) with 76% identity
`
`and 89% similarity to the mouse 6.7 VK gene fragmen
`
`(AF135165). A similar result was obtained using the NCB
`
`lgBlast tool. In this case, two human germline VK sequences
`exhibit good hits with the mouse VK. The closest was the
`
`A17 (X63403) with 76% identity followed by the A18
`
`(X63396) with 74% identity (Lautner-Rieske et al., 1992)
`
`The alignment of the original anti—CD18'VK gene segmen’
`
`l. The
`to the human related sequences is shown in Fig.
`
`A17 coding sequence is identical to the KVZF Swiss-pro
`
`record, while A18 is 81% identical (91% similar) to eithe
`A17 or KV2F. Either VK could be used for grafting the
`
`6.7 VK complementary determinant region (CDR), but the
`
`KV2F/A17 was chosen due to its closer proximity. The
`6.7 J K (AF135165) closest human JK gene segment is the
`
`J K4 with only one difference. Thus the human germline JK4
`
`sequence was chosen for completing the FR4 of the human
`ized VL. The conservative V —> L codon change observed
`
`in the recombinant FR4 (Fig. 1) is due to an Xhol site a
`the end of VL used for the expression vector manipulation:
`
`
`
`3.2. Identification of putative constraints
`
`The visual inspection of two of the closest murine (lMRC
`and human (1AD9) Fab crystals suggests an overall con
`served structure in the VL domain. The systematic survey 0
`
`the tri-dimensional structure at the replacing residues reveal
`
`two positions that could be important for maintaining the
`
`framework structure. The murine residue Leu46 was found
`to be buried in the VH—VL interface. By using a cut—off
`distance of 4.0A, VL residue 46 makes many contacts
`
`
`
`performed with rabbit anti-human lgG-FITC. All samples
`were resuspended in 40011.1 of FACS buffer (PBS, 2%
`FCS and 0.01% sodium azide) and analyzed using a FAC—
`Scan flow cytometer (Becton Dickinson, CA, USA). Ten
`thousand events were analyzed for eaCh sample, inside the
`gate of lymphocytes. Recombinant proteins were added in
`equimolar quantities. Results are expressed as the percent—
`age of stained cells. The antibodies anti~CD4, anti—CD8
`and anti—CD45RO were used in order to characterize the
`
`T—lymphocyte sub-populations that
`were able to bind.
`
`the humanized scFvs
`
`2.8. Blocking capacity of the humanized scFvs.
`
`In order to analyze the binding specificity of the two hu—
`manized scFvs, a blocking experiment was performed. The
`capacity of the scFvs to block the binding of the original
`6.7 anti-CD18 FITC to surface CD18 molecules was tested.
`
`Cells were initially incubated with the two humanized scFvs,
`washed, incubated with rabbit anti—mouse lgG (to block the
`protein A domain present in the recombinant scFvs) and then
`incubated with 6.7 FITC. The percentage of positive cells
`and the intensity of immunofluorescence (IF) were com»
`pared in samples with 6.7 FITC alone and samples with the
`different humanized scFvs plus 6.7 FITC. The percentage
`of inhibition was calculated considering these differences.
`
`3. Results
`
`3.1. Selection of the framework WC and JK for
`CDR-grafting
`’
`
`The human framework used to accept the murine CDRs
`was selected based on the closest germline sequence (Caldas
`
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`VL CD18 6.7
`VK A17
`VLCD18
`VLCD18
`VK A18
`
`(Q)
`(L)
`
`VL CD18 6.7
`
`VK A17|Jk4
`VL CD18
`(Q)
`VL CD18
`(L)
`VK A18
`
`Fig. 1. Design of anti—CD18 VL humanized verSions.’ Amino acid sequence aligmnents of original murine VL (VL 6Z7), closest human germline VL (Vk
`A17 and Vk A18) and humanized VL versions: (VDCDlS (Q) and VLCD18 (L)). CDR residues, according to Kabat et al. (l991) are indicated. Asterisks
`1.,
`.
`indicate the residue 37 and 46, maintained in the‘humanized VL as discussed in the text. Jk residues are shown in italic.
`
`CDR3
`
`
`
`
`
`
`
`4
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`

`

`C. Caldas er £7[./M0/£’('ll[tll' [Immunology 39 (2003) 9414952
`
`945
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`including one to the LCDR 2 residue Phe55, two “Vernier”
`zone residues (Tyr36 and Trp35), framework residues Ile48,
`V3158, and the HCDR3 residues Asp101 e Gly97. There-
`
`fore, we decided to preserve the original murine Leu46 due
`to its array of contacts that includes contacts to VH and
`
`VL CDRs.
`“
`'
`V
`‘
`‘
`,
`Murine residue Leu37- was found to be. superficially buried,
`
`_ making many contacts within the VL dOmain core. Its con-
`- tact residues in lMRC structure at 4.0A cut—off are: Pro44,
`
`_ Lys45, Leu47 and Tyr86. The Lys4L5 contact is one of the
`conserved hydrogen bonds involved in the maintenance of
`the variable domain structure (Chothia et al., 1985). In the
`1AD9 structure, Gln37 contacts the same subset of residues
`as above. The only discrepancy is a hypothetical hydrogen
`bond between the hydroxyl O of the Tyr86 and the amide
`NE of (311137 (d = 282 A). In the lMRC VL, the residue
`37 is partially exposed to the solvent making a contact to a
`water molecule in the crystal. No water was found close to
`residue Gln37 in the VL of 1AD9. A survey of the Kabat
`database showed that position 37 is filled with either glu-
`tamine or leucine. For the mouse light chain, leucine appears
`in 20% of the antibodies while glutamine responds for 78%
`__y of all—mouse antibodies; other amino acid residues make up
`'
`less than 3%. Similar numbers occur for human light chain.
`Due to its location in'the VL-solvent interface and to its
`
`
`
`1:,
`L
`
`close proximity to the CDRl, we chose this position to con-
`struct two humanized versions for the VL domain of the
`
`6.7. The first construction carries the original L37 residue
`(named version L) while the other has the human germline
`Gln37 residue (version Q). Both constructions retained the
`murine Leu46 residue.
`
`3.3. Design of the recombinant humanized VL
`
`The complete humanized VL sequence was obtained by
`grafting the CDRI, 2 and 3 from the 6.7 VL in the proposed
`human germline VK (KV2F/A17) fused to J K4 gene frag-
`ment. CDRl was defined as residues 24 to 34 (24—39 in se—
`quential numbering), CDR2, as residues 50—56 (55—61), and
`CDR3, residues 89—97 (94—102), following the Kabat defi—
`nition (Kabat et al., 1991). The grafting process introduced
`six changes in CDRl, 2 in CDR2 and 5 in CDR3 of the hu-
`man VL, resulting in 13 differences out of the 32 residues
`in the CDRs. The complete humanized sequences are shown
`in Fig. 1. The final proposed humanized sequence had 12
`differences compared to the original 6.7 VL (89% identity)
`and 14 differences to the original VKKVQF/A17/JK4 (87.6%
`identity) in the Leu37 version (Fig. 1). The JK fragment was
`used as is, except for the Leu104 introduced by an XhoI site.
`The proposed recombinant VL was chemically synthesized
`for cloning in an scFv expression vector.
`
`3.4. Construction and expression of the humanized scFv
`
`As shown in Fig. 2, a set of ten .oligomicleotides was
`used to generate the humanized VL ,usgi‘ng'irecombinant PCR
`
`
`
`{"I
`
`
`
`
`
`as previously described for the VH domain (Caldas et al.,
`2000). Briefly, the primers were initially annealed, filled in
`by Klenow and then amplified as pairs until they reached
`the full sized VL (Fig. 2). The synthetic DNA fragment
`was cloned in pGEM—T easy vector. The recombinant clones
`were initially checked for size of insert prior to being repeat-
`edly sequenced. Several clones with the correct sized insert
`were tested and one clone (out of six) of the L version and
`one (out of 8) for Q version had a correct sequence. Correctly
`synthesized DNA inserts were digested with BglII and XhoI,
`and isolated from gel for cloning in the pIg17hVH/mVL
`plasmid cleaved with the same restriction endonucleases.
`This plasmid already harbored a hemi-humanized version
`of the 6.7 anti-CD18 scFv composed of a humanized VH
`fused to the original murine VL (Caldas et al., 2000). The
`cloning procedure eliminates the original murine VL, re—
`placing it with the synthetically humanized gene fragment.
`Two new constructions were obtained: pIg17hVH/hVL(L)
`and pIg17hVH/hVL(Q). For simplicity, these constructions
`were named pIgl7LL and pIgl7LQ, respectively. The whole
`scFv cassette, digested with XmaI/EcoRI, was used to re-
`place the scFv cassette of pPIg16, a P. pastoris expression
`vector (Andrade et al., 2000). According to the notation used
`above, the resulting plasmids were named pPIg LL and pPIg
`~ LQ.
`
`A protease defective strain of P. pastoris was used to re—
`ceive the expression vectors by electroporation. Both plas—
`mids were used to transform electrocompetent yeast cells.
`Many clones were obtained and screened for scFv produc—
`tion in the colony filter assay, where scFv producing cells
`were detected by immunostaining (Andrade et al., 2000).
`Two clones of each construction, found to be positive in the
`filter assay, were selected for growth on an analytical scale.
`In both cases, the detection of recombinant scFv was low.
`
`> Filter assay positive clones were barely visible and the yield
`of purified scFv was also limiting. From 200ml of yeast
`cultures, we normally obtained about 0.5—1mg/l. Even so,
`we were able to purify around 1mg of recombinant scFv
`of both L and Q version of the humanized anti-CD18 for
`further characterization.
`
`3.5. Immunological characterization of recombinant
`humanized antibodies
`
`The CD18 antigen is expressed at the cell surface of all
`leucocytes. Therefore, we tested the two variants of recom—
`binant humanized antibody (LL and LQ) directly for bind-
`ing to peripheral blood mononuclear cells (PBMC) by Flow
`Cytometry analysis. Our results show that the humanized
`LL version presents essentially the same binding capacity
`to lymphocytes as the original anti—CD18 monoclonal anti-
`body, staining 85% of gated cells. In contrast, the LQ ver—
`sion only stained 53% of the cells in the gate of lymphocytes
`(Fig. 3). The original 6.7 anti—CD18 mAb and an anti-DNA
`scFv were used as positive and negative controls, respec-
`tively. The small population stained with high intensity in
`
`5
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`

`

`
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`946
`
`C. Culdus er 11/. /M0[eculcn‘ Immunology 39 (2003) 941—952
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`
`ctt/caa caa aga CCEI
`gaa/gtt g t tCt
`L/Q Q ...................
`
`CCC
`999
`P
`
`L2
`tgc
`
`C t
`
`..........................................................................$.-314011139
`cac acc
`acc
`aac
`aac
`tac
`tc
`ggt
`cac tgg ttt
`cca
`aag
`gtg acc aaa
`F
`H
`W
`
`t99
`
`ttg
`
`ttg
`
`tgg
`
`atg
`
`....................................................................................................................................................
`
`aga
`S
`
`ccg
`G
`
`L6
`gta
`
`gaa
`
`L8
`aga
`tct
`
`gcc
`C99 9
`
`Fig. 2. The VL humanized versions were assembled using a PCR—based protocol. Arrows indicated the sense (—>) and anti-sense (<——) oligonucleoti
`used for VL synthesis. Two variations of the L3 oligonucleotides were synthesized (L3L and L3Q), that create the difference among the versions L and
`
`the humanized versions of anti-CD18 was interpreted as un~
`specific staining, since it was also detected with anti—human
`IgG—FITC alone. The negative control anti-DNA scFV did
`not show any significant staining.
`The humanized scFvs were also able to compete effi-
`ciently with the 6.7 mAb for binding CD18 on lymphocytes,
`as shown in Fig. 4. In this experiment, decreasing dilutions
`of scFV were incubated with PBMC prior to incubation with
`the original anti—CD18 mAb. Both humanized versions were
`able to efficiently block the binding of the mAb to CD18
`molecules at the cell surface. Again, the LL version was
`also more efficient than LQ, blocking up to 80% of rnAb’s
`binding activity in a 1:5 dilution.
`In order to compare the cell subsets recognized by the
`humanized LL version and the original marine antibody,
`we performed FACS analysis, using different markers. We
`found that both the original anti—CD18 and the human~
`ized LL version displayed the same'pattern. of staining
`to CD4+, CD8+ and to memory CD45R‘OTLilymphocy‘tes
`
`(Fig. 5).
`.
`a4;
`* '
`.
`
`
`
`4. Discussion
`
`
`The rules for transferring ligand specificity between donor
`and acceptor antibody are partially known. Today it is co
`
`ceivable to» transfer the CDR regions to a closely related
`framework acceptor, retaining the antibody’s specificity. T ~
`
`fact that the three—dimensional structure of the variable do-
`main is highly conserved makes this strategy feasible. In
`addition, the traditional model of antibody ontogeny relies
`on a flexible variable domain structure for evolving B cell
`response for an infinite antigen repertoire. Somatic hyper—
`mutation molds the final high affinity antibody. Such flexiv
`bility is so impressive that even Ig transgenic mice are able
`to make antibodies successfully even with a very limited 1g
`repertoire (Cascalho et al., 1996). It is likely that antibodies
`are made for binding, and transposing specificity is an opera-
`tion controlled by simple rules. When Wu and Kabat (1970)
`systematically analyzed the primary structure of the variable
`domain of antibodies, they observed that'interspaced in the
`overall variable structure of the amino terminus domain of
`
`
`
`
`
`
`
`
`
`
`
`
`6
`
`

`

`C. Cult/as ct (IL/Molecular Immunology 39 (2003) 941—952
`
`947
`
`FL2—Height
`
`10110210°104
`10° 10
`
`FL2—Height
`
`102103104
`101 10° 10°
`
`(A)
`
`LL + anti—IgG FITC
`
`(B)
`
`LQ + anti—IgG FITC
`
`10210310
`
`FL2—Height
`
`101 10°
`
`10°
`
`101
`
`102
`
`10°
`
`104
`
`FL2-Height
`
`104
`
`102103
`
`
`
`
`104
`10‘
`102
`103
`
`101
`
`
`
`(C)
`
`ZZZ + anti~IgG FITC
`
`(D)
`
`' Anti—CD18 FITC
`
`Fig. 3. The scFv anti~CD18 humanized versions bind to human lymphocyte cell surface; 84, 53 and 85% of the cells were positive when incubated with
`the humanized scFv versions LL (A), LQ (B), or the original monoclonal anti—CD18 antibody (D), respectively. The 222 anti-Z—DNA scFv was used as
`negative control (C).
`
`
`
`
`
`
`
`
`
`both heavy and light chain of antibody, there were hypervari—
`able regions. These regions were termed complementary de—
`termining regions after a new paradigm that implicates this
`region as the determinant for an antibody’s binding speci—
`ficity. This paradigm is still accepted today, and it is the basis
`of CDR-grafting (CDR replacement) as it was initially pro—
`posed by Jones et a1. (1986). From that time on, transferring
`CDRs from mouse antibody to a human antibody framework
`became a general rule for antibody humanization.
`The first humanization procedures were based on trans-
`ferring mouse CDRs to human frameworks derived from
`known human antibodies. Even though this simple opera—
`
`100
`
`80
`
`
`
` ”/0cellsstainedwith67mAbFITC
`
`20
`
`
`0 |
`-i
`—1
`|
`-|
`
`1:5‘
`
`1:10
`
`1:20
`
`1:40
`
`1:80
`
`Dilution of humanized scFvs
`
`Fig. 4. Humanized scFvs block the binding of anti—CD18mAb FITC
`to CD18+ cells. Cells were incubated with different concentrations of
`recombinant humanized seFvs, washed, and then incubated with the mAb
`
`anti-CD18 FITC. The percentage of cells stained With the anti-CD18.mAb
`FITC after the addition of the humanized schvs
`shoving
`‘
`
`
`
`
`
`
`
`+ LL
`+ LQ
`
`
`
`6°
`
`40
`
`tion works, in most cases affinity was lost. To achieve an
`antibody with a better affinity, different authors have applied
`different strategies such as using other human framework V
`genes (Singer et al., 1993), exchanging sterically hindering
`residues (Zhu and Carter, 1995), or replacing residues that
`was known to interfere with CDR conformation (Tempest
`
`,
`
`et al., 1991). Therefore the design of such humanized anti—
`bodies requires correction of the framework.
`_
`In a broader view, most humanized antibodies lose part of
`their affinity compared to the original antibody (Studnicka
`et al., 1994; Tempest et al., 1995', C0 et al., 1996). This
`loss is enhanced as the similarity of the chosen framework
`and the donor variable sequence decreases. Many authors
`elegantly demonstrated this framework effect (Tramontano
`et al., 1990; Foote and Winter, 1992; Studnicka et al., 1994).
`Tramontano et al. (1990) demonstrated the effect of the
`residue 71 heavy chain as a prototype of framework bias on
`I the CDR conformation. Many other trending framework po—
`sitions were revealed by systematically analyzing antibody
`X—ray crystals (Foote and Winter, 1992; Studnicka et al.,
`1994). Thus, an influence of the framework in paratope
`assembling became widely accepted. It may appear con-
`tradictory because CDRs are the most variable portion of
`the variable domain, but recent studies bring evidence that
`CDRs indeed determine the overall shape of the paratope
`(Holmes and Foote, 1997; Holmes et al., 1998). The best
`scene is of a framework that sustains the paratope but may
`constrain certain conformational spaces. CDR would shape
`the paratope and bias the whole variable region structure in—
`side the framework intrinsic limitations.
`
`7
`
`

`

`948
`
`C. C(Ildas e! (1/. /Molecular Immunology 39 (2003) 941—952
`
`mAb anti-CD18
`
`Humanized LL
`
`FL2~Height
`
`10210310“
`101
`
`
`3 ..
`:3a.
`102
`10
`FL 1-Height
`
`1a“
`
`FL2~Height101102103104
`
`10° 34:1
`
`10
`
`102
`FL1~Height
`
`1o
`
`1
`
`
`
`
`
`10310“
`FL2-Height 102
`
`1a1
`
`10‘I
`
`m
`
`1132
`FL'l-Height
`
`in
`
`m“
`
`CD4+
`
`FL2—Height
`
`104
`
`103
`
`102
`
`in“
`
`o1
`
`1o
`
`1o
`FLI-Height
`
`10
`
`1o
`
`10‘
`10°
`
`
`11:1311:14
`
`FL2-Height 102
`
`10‘
`
`FL1-Height
`
`103
`
`FLZ-Height 102
`
`CD8+
`
`CD45RO+
`
`
`
`
`
`
`
`
`CD18+
`
`,
`102
`FL1-Hei ht
`
`m
`
`Fig. 5. T cell subpopulations stained by the humanized LL version compared to the original mAb.
`
`In this report we synthesized the light chain variable re—
`gion as part of a humanized anti-human CD18 antibody. The
`strategy to design the humanized light chain variable region
`was based on the closest germline sequence (Caldas et al.,
`2000). Such a strategy allows us to reduce the framework
`derived constraints due to the use of the closest germline
`
`framework sequence. Many human germline sequences are
`available to date, facilitating the search for hOmologous V
`gene candidates (Tomlinson et al., 1995). After design we
`proceeded to the visual inspection of the three-dimensional
`structure of similar antibodies. In this step, we chose to use
`
`X-ray derived information instead of the molecular model
`for the humanized structure, because there is a large amount
`
`of data on crystal structures of antibodies in the PDB,
`(This is probably the most studied protein family at the
`three—dimensional level ) We were able to find, in the PDB,
`two sequences which were very similar to our VL sequence;
`one is a mouse Fab and the other is a humanized Fab
`There are 12 differences between themurine and the hu-
`manized VL Most of them correspond to exposed residues
`
`
`or residues far from the paratope. There was one importa
`residue, Leu46, that was shown to make contacts to LCDR _
`and 3, besides playing an important role in the interface of
`VL/VH, making close contact to HCDR3 (Padlan, 1994):
`The exchange of a leucine, an aliphatic amino acid, for the
`arginine that appears in the human closest germline, would
`probably disrupt many atomic contacts and destabilize the
`overall structure. Therefore this residue was kept as in the
`original murine antibody.
`Another observed difference was at position 37. This .:
`residue sits in the bottom of the molecule between the struc-
`turally conserved Gln38, involved in light chain—heavy chain f
`interface (Chothia et al., 1985), and the “Vernier” zone _
`residues after CDRl (Foote and Wintei 1992). In the mouse 5
`antibody structure lMRC, the Leu37 is partially accessible ‘
`to solvent where it is close to a water molecule (3. 37 A)
`It is probably not involved in a direct H-bond since the
`distance found is over the expected distance of a regular
`water—carbonyl hydrogen bond (CreigthOn, 1984). In the hu—
`man VK germline sequence used in this work, a glutamine
`
`
`
`-
`_
`
`
`
`8
`
`

`

`C. Caldas er (IL/Molecular Inunmm/Ugy 39 (2003) 9417952
`
`949
`
`
`
`
`
`
`
`
`" Fig. 6. Structural visualization of the Gln37—Tyr86 interaction. The structure of variable domain of 1AD9 was visualized using RASMOL. VL backbone
`is shown in gray and its three CDRs (black) are labeled. Gln37 and Tyr86 residues are shown in a space filling representation. The atoms labeled with N
`and 0 denotes the glutamine amide group and the tyrosine hydroxyl group, respectively. Heavy chain backbone (light gray) is shown in the background.
`
`
`
`residue fills the position 37. This residue is found in many
`2 humanized antibodies, including our closest PDB crystal
`, structure, 1AD9. In this