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The Journal of Immunology, 1998, 160: 1029-1035.
Copyright © 1998 by The American Association of Immunologists

Humanization and Pharmacokinetics of a Monoclonal Antibody with Specificity for Both E- and P-Selectin

Xing-Yue He, Zhenghai Xu, Jennifer Melrose, Alison Mullowney, Maximiliano Vasquez, Cary Queen, Vladimir Vexler, Corine Klingbeil, Man Sung Co and Ellen L. Berg1

Protein Design Labs, Inc., Mountain View, CA 94043


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
E- and P-selectin (CD62E and CD62P) are cell adhesion molecules that mediate leukocyte-endothelial cell and leukocyte-platelet interactions and are involved in leukocyte recruitment during inflammation. We previously developed a murine mAb, EP-5C7 (or mEP-5C7), that binds and blocks both E- and P-selectin. When used in humans, murine mAbs have short circulating half-lives and generally induce potent human anti-mouse Ab responses. We therefore engineered a humanized, complementarity determining region-grafted version of mEP-5C7 incorporating human {gamma}4 heavy and {kappa} light chain constant regions (HuEP5C7.g4). HuEP5C7.g4 retains the specificity and avidity of mEP-5C7, binding to human E- and P-selectin but not to human L-selectin, and blocking E- and P-selectin-mediated adhesion. Surprisingly, when administered to rhesus monkeys, HuEP5C7.g4 was eliminated from the circulation very rapidly, even faster than the original murine Ab. To isolate the cause of the short serum half-life of HuEP5C7.g4, several Ab variants were constructed. A chimeric IgG4 Ab was made by replacing the humanized V regions with murine V regions. A humanized IgG2 Ab, HuEP5C7.g2, was also made by replacing the human {gamma}4 with a {gamma}2 constant region. Results from pharmacokinetic studies in rhesus monkeys demonstrated that the chimeric IgG4 is also rapidly eliminated rapidly from serum, similar to the humanized IgG4 Ab, while the humanized IgG2 Ab displays a long circulation half-life, typical of human Abs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The selectin family consists of the L-, E-, and P-selectins expressed on leukocytes, activated endothelial cells, and activated platelets. The three selectins act in concert with other cell adhesion molecules to permit recruitment of leukocytes from the blood into inflamed tissues (1, 2). Current models suggest that the selectins mediate initial adhesive rolling of leukocytes on the endothelial vessel wall, a process followed by firm attachment and transmigration of leukocytes, which are mediated by other adhesion molecules.

Abs that block E- or P-selectin inhibit the inflammatory sequelae in several animal models of inflammation, including leukocyte-mediated tissue damage due to ischemia/reperfusion injury (3, 4, 5, 6, 7, 8, 9). Results from studies in which Abs to E- and P-selectin are used in combination and related studies in knockout mice have suggested that E- and P-selectin can work cooperatively together in some inflammatory conditions, and the absence of one may be compensated by the other (10, 11, 12, 13). For therapeutic treatment of these inflammatory conditions, blockade of both selectins is preferred and could be provided by a cross-reactive mAb, such as mEP-5C7, that binds and blocks both E- and P-selectin (14).

Direct use of murine mAbs for human therapy is limited by the development of an immune response by the recipient patient against mouse-specific antigenic determinants, i.e., human anti-mouse Ab responses, as well as the presence of unwanted or the lack of desired human effector functions on mouse Abs. The human anti-mouse Ab response can result in potentially harmful reactions and the rapid clearance of circulating Ab (15, 16, 17). To reduce the immunogenicity of mouse Abs for human therapy, chimeric Abs were initially developed. Chimeric Abs retain the mouse V regions, and hence the specificity, of the parental rodent Abs, but replace the constant regions with the appropriate human heavy and light chain sequences. The use of chimeric Abs clearly addresses the issue of immunogenicity of the constant region caused by species differences, but V region differences remain a potential source of immunogenicity (18, 19).

Humanized Abs are generated by grafting complementarity-determining regions (CDRs)2 of mouse Abs into a background of human framework regions and constant regions by recombinant DNA technology (20). Humanized Abs contain only ~10% mouse sequences and 90% human sequences, and thus further reduce or eliminate the immunogenicity while retaining the Ab specificities (21, 22). Using a computer-aided CDR-grafting method, we have successfully humanized several mouse Abs for potential treatment of various human diseases (23, 24, 25, 26, 27). Several humanized Abs, including humanized M195 (anti-CD33, a myeloid leukemia surface Ag) and humanized anti-Tac (anti-IL-2 receptor {alpha} subunit), have been evaluated in human clinical trials, demonstrating long half-lives and no evidence of immunogenicity when administered in multiple doses (24, 28). In this report we describe an unexpected outcome of the humanization of mEP-5C7; when injected into rhesus monkeys, the humanized Ab, HuEP5C7.g4, displayed a very short circulating half-life. A greatly lengthened circulating half-life was obtained, however, by replacement of the human IgG4 heavy chain constant region with a human IgG2 constant region.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and cell lines

Mouse monoclonal anti-human E/P-selectin EP-5C7, anti-human P-selectin WAPS 12.2, anti-human E-selectin E-1E4, and anti-human CD18 NA8 Abs have been described previously (14). Mouse (IgG1 {kappa}) myeloma MOPC 21 and purified human (IgG4 {kappa}) were obtained from Sigma Chemical Co. (St. Louis, MO). SP2/0-Ag14, a mouse myeloma cell line, and HL60, a human promyelocytic cell line, were obtained from American Type Culture Collection (CRL 1581, and CCL 240, Rockville, MD). A mouse pre-B cell line, L1-2, transfected with human P-selectin, L1-2P-selectin, and Chinese hamster ovary (CHO) cell lines transfected with human E- or P-selectin gene (CHOE-selectin or CHOP-selectin cells) were described previously (14). HuDREG-200, is a humanized IgG4 Ab (29), and binds human, but not rhesus, L-selectin (E. Berg, unpublished observations). D200Id and 2H9 are mouse monoclonal anti-Id Abs generated against HuDREG-200 and HuEP5C7.g4, respectively. The hybridoma producing 2H9 was generated by injecting BALB/c mice in the hind footpads with 10 µg of purified HuEP5C7.g4 prepared in monophosphoryl lipid A plus trehalose dicorynomycolate adjuvant (Sigma Chemical Co.), then 4 and 7 days later with Ab in PBS. Three days following the last immunization, fusion with P3X cells was performed, generally as previously described (14). Hybridoma supernatants were screened by ELISA on plates coated with HuEP5C7.g4 in the presence of 10% normal human serum (Sigma Chemical Co.). 2H9 binds HuEP5C7.g4 and mEP-5C7, but fails to bind a panel of other humanized Abs or purified human IgG4.

Cloning of V region cDNAs

The V domain cDNAs of the light and heavy chains of mEP-5C7 were cloned by the anchored PCR method using 3' primers that hybridize with the C regions and 5' primers that hybridize with the G tails attached to the cDNA using terminal deoxytransferase (30). The sequences were determined using the dideoxy termination method with an Applied Biosystems 373A automated sequencer (Foster City, CA).

Computer analysis

Three-dimensional models of the variable domains of the mEP-5C7 Ab were built with the help of the ABMOD and ENCAD programs created by Levitt and co-workers (31, 32). Sequence analysis, homology searches, and structural analysis of three-dimensional models were conducted with the program DELTA developed in our laboratory (33).

Construction of chimeric and humanized Abs

See Figure 3Go for a description of the Abs constructed. Plasmids pV{kappa}, containing the human Ck region (25, 34), and pVg4, containing the human {gamma}4 constant region, were used for the construction of chimeric and humanized versions of mEP-5C7 of the human IgG4 isotype. pVg4 is identical with pVg1 except for containing heavy chain sequences from human {gamma}4 instead of {gamma}1 (25). The XbaI/BamHI fragment containing the human {gamma}4 constant region in the pVg4 expression vector was derived from phage provided by L. Hood (35). For the construction of humanized HuEP5C7.g2, the plasmid pVg4 was replaced by pVg2.M3.D.Tt (36). pVg2.M3.D.Tt incorporates two mutations into a human {gamma}2 constant region (with Ala replacing Val and Gly at positions 234 and 237, using the EU numbering system) to reduce interaction of IgG2 with Fc receptors and minimize Ab effector activity. Heavy and light chain expression plasmids were constructed by inserting the appropriate V region genes into the XbaI site of the respective vectors.



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  		FIGURE 3. Humanized and murine Abs. The species, isotype, and type of V region heavy and light chains for each Ab are listed. A representational figure for each Ab is also shown. Regions containing murine sequences are shown in black, human {kappa} light chain constant regions (C{kappa}) are gray with black outlines, humanized VL and VH are white, human IgG4 heavy chains are horizontally striped, and human IgG2 heavy chains are gray.

 
For the construction of chimeric Ab expression vectors, XbaI fragments containing the murine VL and VH genes (including the 5' signal peptide sequence and the 3' splice donor signal) were constructed by PCR from their respective murine V region cDNAs and then inserted into the XbaI site of the respective pV{kappa} and pVg4 vectors. For the construction of humanized Ab expression vectors, XbaI fragments containing the humanized VL and VH genes (including the 5' signal peptide sequence and the 3' splice donor signal) were constructed from assembling eight synthetic oligonucleotides.

Nucleotide sequences were designed to encode the protein sequences of the humanized EP5C7 VL and VH, generally using codons found in the mouse sequence. Several degenerate codons were changed to create convenient restriction sites or remove undesired ones. To synthesize each V gene, four pairs of oligonucleotides (~80 bases in length) were synthesized (380B DNA Synthesizer, Applied Biosystems) with overlapping stretches of 20 nucleotides. Assembly and amplification of the genes were conducted in four steps: 1) the four pairs of complementary oligonucleotides were annealed and extended with Klenow fragment in separate reactions; 2) the resulting four dsDNA fragments were mixed pairwise, denatured, reannealed, and extended in two separate reactions; 3) the resulting two dsDNA fragments were mixed, denatured, reannealed, and extended to create the final full-length dsDNA; and 4) the resulting DNA was used for PCR amplification with two short primers, which correspond to the 5' and 3' ends of the template DNA and contain an XbaI site for subsequent cloning.

The synthesized XbaI fragments were then inserted into the XbaI site of the respective pV{kappa}, pVg4, or pVg2.M3.D.Tt vectors. Hybrid Abs containing a humanized {gamma}4 heavy chain and chimeric light chain (HuH/ChL) or a chimeric {gamma}4 heavy chain and a humanized light chain (ChH/HuL) were also constructed for evaluation.

Production and purification of humanized and chimeric Abs

Cells producing humanized Abs (HuEP5C7.g4 or HuEP5C7.g2), chimeric Abs (ChEP5C7.g4), or hybrid Abs (Hybrid HuL/ChH, or Hybrid HuH/ChL) were generated by transfection of Sp2/0 cells with the appropriate plasmids by electroporation using a Gene Pulser apparatus (Bio-Rad, Hercules, CA) at 360 V and 25 µF capacitance according to the manufacturer’s instructions. Before transfections, plasmids were linearized using PvuII. All transfections were performed using 20 µg of plasmid DNA and 107 cells in PBS. The cells from each transfection were plated into four 96-well tissue culture plates. After 48 h, selective medium was applied. Cells were selected in DMEM/10% FBS/hypoxanthine/thymidine media supplement (Sigma Chemical Co.) and 1 µg/ml mycophenolic acid. Ab-producing clones were screened by assaying for the presence of Ab in the culture supernatant by ELISA. Abs from transfectants as well as hybridomas were purified from serum-free culture supernatants (Hybridoma-SFM, Life Technologies, Gaithersburg, MD) by passage over a column of staphylococcal protein A-Sepharose CL-4B (Pharmacia, Piscataway, NJ). The bound Abs were eluted with 0.2 M glycine-HCl, pH 3.0, and neutralized with 1 M Tris-HCl, pH 8.0. The buffer was exchanged for PBS by passing over a PD10 column (Pharmacia).

Avidity measurement

Binding avidities of mEP-5C7 and HuEP5C7.g4 with E- and P-selectin were determined by competitive binding of radiolabeled Ab to CHOE-selectin and L1-2P-selectin cells, respectively. Purified mEP-5C7 Ab was labeled with Na125I (Amersham, Arlington Heights, IL) using lactoperoxidase at 4 mCi/mg of protein (37). Increasing amounts of cold competitor Ab (mEP-5C7 or HuEP5C7.g4) were added to 2 ng of radiolabeled tracer mEP-5C7 Ab and incubated with 4 x 105 CHOE-selectin or L1-2P-selectin cells in 0.2 ml of binding buffer (PBS with 2% FCS and 0.1% sodium azide) for 2 h at 4°C with constant shaking. Cells were washed and pelleted, and the radioactivity associated with the cell pellets was measured. The ratio of bound and and free tracer Ab was calculated, and the avidities were calculated according to the formula: [X] - [EP5C7] = (1/Kx) - (1/Ka), where Ka is the avidity of EP5C7, Kx is the avidity of the competitor X, brackets indicate the concentration of competitor Ab at which bound/free tracer binding is R0/2, and R0 is maximal bound/free tracer binding (38).

E- and P-selectin adhesion assays

HL-60 cell binding to CHOE-selectin or CHOP-selectin transfectant cell lines was performed as described previously (14). Confluent cultures of CHOE-selectin or CHOP-selectin transfectant cells, grown in 96-well plates, were washed and incubated for 15 min with 50 µl of assay buffer (10% adult bovine serum/10% normal rabbit serum/10 mM HEPES (pH 7.2)/RPMI) containing various test and control Abs (Sigma Chemical Co.) at 10 µg/ml. Fluorescently labeled HL-60 cells were prepared as previously described (14) and resuspended in assay buffer containing 0.25 µg/ml anti-CD18 Ab, NA8, at 2 x 106 cells/ml. Assays were initiated by addition of 50 µl of HL-60 cells to CHOE-selectin or CHOP-selectin cells for a final volume of 0.1 ml while plates were rotated at 40 rpm (Innova 200 orbital shaker, New Brunswick, Inc., Edison, NJ). After 15 min at room temperature, unbound cells were removed by washing plates four times with 0.2 ml RPMI/well. Bound cells were fixed to the CHOE-selectin or CHOP-selectin cells by addition of 100 µl 1% paraformaldehyde (Sigma) in PBS. Plates were analyzed using a Microplate Fluorometer (model 7620, Cambridge Technology, Inc., Watertown, MA), and the relative number of cells bound per well was calculated from the total amount of fluorescence measured at 530 nm, using an excitation at 485 nm.

Pharmacokinetics of murine and humanized Abs in rhesus monkeys

Pharmacokinetic studies were performed at the California Primate Research Center (Davis, CA). Rhesus monkeys (3–5 kg; three per group) were injected with 2 mg/kg of the indicated Abs. Serum samples were collected relative to Ab injection at -7 days, 0 (predose), 0.5, 1.5, 2, 6, 12, and 18 h and 1, 2, 3, 4, and 7 days. Samples from some animals were also obtained on days 14, 15, and/or 21.

Pharmacokinetic analysis

Compartment-independent pharmacokinetic analysis (39) was performed using the KaleidaGraph program for Macintosh (Developed by Abeldeck Software, distributed by Synergy Software, Reading, PA). The area under the concentration-time curve, AUC0-tlast, was calculated by the trapezoidal rule. AUCtlast-{infty} was determined by extrapolating to infinity from Ctlast assuming exponential decay using the equation AUCtlast-{infty} = Ctlast/k2, where Ctlast is the concentration at the last measured time point, and k2 is the elimination rate constant of the terminal portion of the concentration-time curve. AUC0-{infty} was derived from AUC0-tlast + AUCtlast-{infty}. The parameter k2 was estimated by linear regression analysis of the terminal portion of the curve from a minimum of four last data points. The elimination half-life, t1/2, was derived from 0.693/k2.

ELISAs

Microtiter plates (Nunc Immunolon 1) were coated overnight with 100 µl/well of capture Abs. These included polyclonal anti-mouse IgG (H+L) (human Ig absorbed, Biosource, Camarillo, CA), diluted 1/200, or anti-Id Abs against EP5C7 (2H9) or HuDREG-200 (D200Id) at 2 µg/ml in PBS. Plates were then blocked by incubating wells with 0.5% nonfat dry milk/PBS for 1 to 2 h and subsequently washed twice in 0.05% Tween-20/PBS. Serum samples diluted appropriately (1/10 to 1/2000) or standards diluted and prepared in 0.5% milk/PBS were added to wells, and plates were incubated overnight at 4°C. Following four washes in 0.05% Tween-20/PBS, secondary Abs were applied. These included horseradish peroxidase (HRP)-conjugated Fc-specific anti-mouse Ig (The Jackson Laboratory, Bar Harbor, ME) at a 1/4000 dilution for evaluation of mEP-5C7, HRP-conjugated anti-human IgG4 (The Binding Site, Birmingham, U.K.) at a 1/1000 dilution for evaluation of HuEP5C7.g4, and chimeric and hybrid IgG4 Abs and HRP-conjugated anti-human IgG2 (The Binding Site) at a 1/400 dilution for evaluation of HuEP5C7.g2. After 1 h, plates were again washed in 0.05% Tween-20/PBS. Bound Abs were detected by addition of 100 µl/well TMB substrate (Kirkegaard and Perry, Gaithersburg, MD), color development for 10 min, termination of the reaction by addition of 100 µl/well 2 M H2SO4, and then measurement of absorbance at 450 nm.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning and sequencing of V region cDNA

The murine EP-5C7 Ab heavy and light chain V region cDNAs were amplified using an anchored PCR method (30), then cloned into pUC18 for sequence determination. Nucleotide sequences were obtained from several independent clones for both VL and VH cDNAs. For the heavy chain, a unique sequence was identified. For the light chain, two sequences were identified. One sequence had a nucleotide missing at the end of the V gene, causing a frame shift at the V-J junction, and was identified as the nonproductive allele from the fusion partner cells. The other sequence is typical of a mouse {kappa} chain V region. The nucleotide and translated amino acid sequences of the coding light and heavy chain V regions of mEP-5C7 are shown in Figure 1Go. Sequence analysis indicates that the light chain V region gene of mEP-5C7 belongs to mouse {kappa}-chain subgroup I, and the heavy chain gene belongs to mouse heavy chain subgroup III (40).



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  		FIGURE 1. cDNA and translated amino acid sequences of the heavy chain (A) and light chain (B) V regions of the murine EP-5C7 Ab. The three CDRs in each chain are underlined. The first amino acid of the mature light and heavy chains are double underlined, preceded by their respective signal peptide sequences.

 
Modeling and design of humanized sequences

For humanization, the approach of Queen et al. (23) was followed. First, sequences of human V regions most similar to mEP-5C7 were identified. Among the best VH sequences is III-3R (41) of Kabat’s subgroup III, with 72% identity in the framework region. The VL from III-3R was also used. This VL belongs to the Kabat subgroup I of {kappa}-chains and has 61% identity with mEP-5C7 in the framework region.

With the help of the three-dimensional model, a number of framework positions were identified that differed between mEP-5C7 and the chosen human III-3R sequence and whose location in three-dimensional space relative to the hypervariable regions, or CDRs, makes it likely that they could influence CDR conformation, and thus binding affinity. Amino acids from the murine EP5C7 sequence were used in those positions. This was performed at residues 69 and 70 in the VL domain, but was not required at any residues in the VH domain. Furthermore, in agreement with the protocol outlined previously (23), a number of amino acids in the III-3R human sequences that were unusual at their positions for their respective (human) subgroups were also identified. Those amino acids were changed to correspond to consensus human sequences of human subgroup III (VH) or subgroup I (VL). This was performed at residues 61, 72, 82, and 99 of VL and at residues 1, 75, and 78 of VH. The final amino acid sequences of the humanized EP-5C7 heavy and light chain V regions are shown compared with the murine sequences in Figure 2Go.



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  		FIGURE 2. Amino acid sequences of the mature heavy chain (A) and light chain (B) V regions of the humanized EP-5C7 (lower lines) and the mouse EP-5C7 (upper lines) Abs. The three CDRs in each chain are underlined once. Residues in the framework that have been replaced with mouse amino acids are also underlined once, and consensus human amino acids, different from those in III-3R, in the humanized Ab are doubly underlined.

 
Avidity measurement

Recombinant Abs containing mouse or humanized V regions and mouse or human constant regions, as shown in Figure 3Go, were produced as described in Materials and Methods. The avidities of the humanized Ab, HuEP5C7.g4, for E-selectin and P-selectin were determined and compared with those of mEP-5C7 by competition with the radioiodinated mEP-5C7 Ab. CHOE-selectin cells and L1-2P-selectin cells (14) were used as sources of E-selectin and P-selectin. The binding avidities were calculated as described in Materials and Methods. HuEP5C7.g4 had an avidity of 3.3 x 108 M-1 for E-selectin, identical with that measured for mEP-5C7, while for P-selectin, HuEP5C7.g4 had an avidity of 1.5 x 108 M-1, and mEP-5C7 had an avidity of 6.7 x 108 M-1.

Blocking of E-selectin- and P-selectin-mediated adhesion

To compare the abilities of HuEP5C7.g4 and HuEP5C7.g2, the humanized EP5C7 Ab of IgG2 isotype, with that of mEP-5C7 to inhibit binding of E-selectin to its counter-receptor, these Abs were tested for their ability to block binding of HL-60 cells to E-selectin transfectant cells. Assays of the adhesion of HL-60 cells with CHOE-selectin cells were performed as previously described (14) in the presence of the various Abs at the indicated concentrations. Figure 4Go (A and B) shows that both HuEP5C7.g2 and HuEP5C7.g4 blocked binding of HL-60 cells to CHOE-selectin transfectants as well as or slightly better than mEP-5C7.



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  		FIGURE 4. Inhibition of HL-60 cell adhesion to CHOE-selectin or CHOP-selectin cells by mEP-5C7 ({square}), HuEP5C7.g2 ({blacksquare}), or HuEP5C7.g4 ({triangleup}) Abs. Fluorescently labeled HL-60 cells were incubated with CHOE-selectin (A and B) or CHOP-selectin (C and D) cells in the presence of mEP-5C7 ({square}) and HuEP5C7.g2 ({blacksquare}; A and C) or HuEP5C7.g2 ({blacksquare}) and HuEP5C7.g4 ({triangleup}; B and D) at the indicated concentrations for 15 min. After washing, the relative number of bound cells was determined as described in Materials and Methods. The results from representative experiments performed with each sample in quadruplicate (± SD) are shown.

 
To measure the abilities of HuEP-5C7.g2, HuEP5C7.g4, and mEP-5C7 to inhibit binding of P-selectin to its counter-receptor, the binding of HL-60 cells to CHOP-selectin transfectants in the presence of these Abs was determined. Figure 4Go (C andD) shows that HuEP5C7.g2 and HuEP5C7.g4 block binding of HL-60 cells to P-selectin transfectants as well as mEP-5C7. Isotype-matched control Abs had no effect on binding in this assay (14) (data not shown).

Pharmacokinetics in rhesus monkeys

The elimination profiles for 1 wk following injection in rhesus monkeys of mEP-5C7, HuEP5C7.g4, HuEP5C7.g2, chimeric or hybrid IgG4 Abs, or a control humanized IgG4 Ab, HuDREG-200, that does not bind to any Ag in rhesus monkeys are shown in Figure 5Go. HuEP5C7.g4 and chimeric and hybrid IgG4 versions of EP5C7 all cleared rapidly from the circulation, so their serum levels were <1 µg/ml after 72 h, while HuEP5C7.g2 displayed a long serum half-life, with a level of >10 µg/ml at 72 h. The calculated terminal elimination half-lives calculated from the last several time points and averaged for three rhesus monkeys are shown in Table IGo. The terminal elimination half-life calculated for HuEP5C7.g2 (6.6 ± 0.8 days) was similar to that for the control humanized IgG4 Ab, HuDREG-200 (6.5 ± 1.2 days). However, while the half-life calculated for mEP-5C7 was as short as the half-lives calculated for the IgG4 versions of EP5C7 (2.5 ± 0.7 vs 2.2–2.9 days), inspection of Figure 5Go shows that mEP-5C7 behaves more like HuEP5C7.g2 and the control humanized IgG4, HuDREG-200, up until day 4. The 2-wk elimination profiles for each of three individual rhesus monkeys given HuEP5C7.g2 at 2 mg/kg are shown in Figure 6Go.



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  		FIGURE 5. One-week pharmacokinetic profiles of murine, humanized, and chimeric Abs in rhesus monkeys. Rhesus monkeys (three per group) were given 2 mg/kg of each of the following Abs: mEP-5C7, HuEP5C7.g4, HuEP5C7.g2, HuDREG-200 (control HuIgG4), or chimeric (ChEP5C7.g4) or hybrid (Hybrid HuL/ChH or Hybrid HuHChL) EP5C7 Abs with human IgG4 constant regions. At the indicated times, serum samples were prepared and analyzed for the presence of the indicated Ab as described in Materials and Methods. The mean and SD of the serum concentration of Ab measured in three animals are shown.

 

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  		Table I. Terminal elimination half-lives in rhesus of murine and humanized Abs tested1

 


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  		FIGURE 6. Two-week pharmacokinetic profiles of HuEP5C7.g2 in rhesus monkeys. Three rhesus monkeys were given 2 mg/kg of HuEP5C7.g2. At the indicated times, serum samples were prepared and analyzed for the presence of HuEP5C7.g2 as described in Materials and Methods. The mean and SD of the amount of Ab measured in triplicate serum samples taken from individual rhesus monkeys are shown. The pharmacokinetic parameters shown were determined by analyzing the 3-wk elimination profiles of HuEP5C7.g2 and HuDREG-200 and the 1-wk elimination profiles of other Abs in rhesus monkeys (three per group) given 2 mg/kg. Data were analyzed as described in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously described the identification of a mAb that binds and blocks the adhesive functions of both E- and P-selectin (14). Blockade of these receptors may have clinical utility in situations of leukocyte-mediated tissue damage, such as ischemia/reperfusion injury (myocardial infarction or stroke), trauma, and shock, as well as chronic inflammatory conditions, such as rheumatoid arthritis or psoriasis. For therapeutic purposes, Abs have certain advantages over conventional small molecule drugs, e.g., relatively long serum half-lives and often more attractive safety profiles. Abs are bivalent, and the relatively spacious Ag-combining site naturally provides for a number of Ag contact points. Furthermore, the ability to "humanize" high affinity murine mAbs has resulted in improved therapeutic efficacy of Abs by reducing immunogenicity and improving serum half-lives.

The techniques of Ab humanization have evolved over the past 9 yr, so that in our laboratory, humanization of murine Abs specific for single protein Ags is nearly always successful. The case described here, however, is the first report of the humanization of an Ab with reactivity against two Ags, the E- and P-selectins. Although these two proteins share homology, there are significant differences between them (the various domains share 34–62% amino acid identity) (42, 43). This additional constraint made the likelihood of success of the humanization process less predictable.

Humanization of mEP-5C7 was successful in that the avidity of the humanized Ab, HuEP5C7.g4, for both E- and P-selectin was substantially retained. Interestingly, the murine Ab slightly favors P-selectin, while HuEP5C7.g4 slightly favors E-selectin. These slight differences in avidity are not likely to affect the efficacy of the humanized Ab in vivo, since they are not accompanied by changes in functional blocking activity (Fig. 4Go).

Since mEP-5C7 and HuEP5C7.g4 bind to the E- and P-selectins of nonhuman primates, including rhesus and baboon (44) (data not shown), pharmacokinetic studies were performed in both species. Unexpectedly, HuEP5C7.g4 displayed a very short elimination half-life, clearing from the circulation in both rhesus and baboons even more rapidly than mEP-5C7, particularly in the first 4 days (Fig. 5Go) (V. Vexler, unpublished observations). Several possibilities could account for a more rapid elimination profile of a humanized Ab than the original murine Ab, including chemical or physical instability, or creation of a novel binding site for another Ag. Such a novel binding site might have specificity for a single widely expressed Ag or might be nonspecific, binding many Ags but with low affinity (as do some natural Abs) (45).

HuEP5C7.g4 exhibited no obvious physical or chemical instability (data not shown), nor did it bind nonspecifically to Ags (e.g., BSA, insulin, and DNA) in vitro (not shown). In addition, tissue cross-reactivity studies on baboon tissues did not identify any unexpected tissue reactivity of HuEP5C7.g4 (V. Vexler, unpublished observations). To identify the probable cause of the rapid elimination of HuEP5C7.g4, four additional Abs were generated: a chimeric Ab (mouse VL and VH), two hybrid Abs (mouse VL and humanized VH, or humanized VL and mouse VH) with human IgG4 constant regions, and a humanized Ab of the human IgG2 isotype, HuEP5C7.g2.

The serum half-lives of chimeric and hybrid Abs in rhesus monkeys were short, similar to that of HuEP5C7.g4 (Fig. 5Go). In contrast, changing the isotype of the constant regions from IgG4 to IgG2 resulted in a humanized Ab with a longer serum half-life (Figs. 5Go and 6Go). This Ab, HuEP5C7.g2, was identical with HuEP5C7.g4 in its specificity for E- and P-selectin and its ability to block E- and P-selectin-dependent adhesion in vitro (Fig. 4Go).

The short half-life of HuEP5C7.g4 does not appear to be due to the {gamma}4 constant region per se, because the control Ab HuDREG-200 of the IgG4 isotype has a significantly longer half-life (Fig. 5Go and Table IGo). Although HuDREG-200 was the only control IgG4 Ab tested, the 6.5-day terminal elimination half-life obtained in rhesus monkeys is likely to be in the normal, expected range and not aberrantly long. In humans, metabolic turnover studies with myeloma IgG4 Abs suggest that IgG4 Abs have half-lives comparable to those of IgG1 Abs (46). The reported half-lives of human and humanized IgG1 Abs in rhesus monkeys are similar and range from 9 to 30 days (47, 48). However, the short half-life of HuEP5C7.g4 cannot be due only to its V region either, because when the same humanized V region is combined with a human {gamma}2 constant region in HUEP5C7.g2, a long half-life results. Moreover, the murine Ab mEP-5C7, with a V region of the same specificity and affinity as HuEP5C7.g4, has a half-life as long as can be expected for mouse Abs injected into primates. It is only the combination of the EP-5C7 V region binding site in either murine or humanized form together with the human {gamma}4 constant region, whether in HuEP5C7.g4, the chimeric ChEP5C7.g4 Ab, or the hybrid humanized/chimeric Abs, that leads to rapid elimination from the serum (Fig. 5Go).

Possible explanations for these findings are 1) the mode of flexibility of the human {gamma}4 hinge region may allow the EP-5C7 V region to bind with some affinity to other Ags in rhesus monkeys (perhaps to other lectin-like domains), leading to rapid clearance from the serum, or 2) the ability of the {gamma}4 constant region to bind to certain Fc receptors on leukocytes together with the ability of the V region to bind to E- and P-selectin may create an interaction that leads to clearance of the Ab from the serum. The mutated {gamma}2 region used here, which was engineered to eliminate binding to Fc receptors (36), would avoid this problem. These are but two of the number of potential explanations left after more obvious ones were ruled out by our experimental results. The rapid clearance of HuEP5C7.g4 from the serum is more likely due to Ab sequestration and not elimination from the body, since the rate of clearance of HuEP5C7.g4 observed during the first several days after administration slows down significantly after 4 days (Fig. 5Go).

Regardless of the reason for the short-half life in primates of HuEP5C7.g4, the availability of the humanized HuEP5C7.g2 Ab with its unique function of binding and blocking both E- and P-selectin and with a long half-life in the circulation presents a viable anti-inflammatory drug candidate for clinical evaluation.


   Acknowledgments
 
Sincere thanks to Lisa Hernandez and Chuck Bullock for organizing the pharmacokinetic studies, to Heather Sternard and Paul Sauer for cell culture, to Brad Tada and Julie Mikkelsen for Ab purification, and to Brent Larsen, Mike Klein, and Eddie Kast for protein analysis to confirm the identity of the recombinant Abs.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Ellen L. Berg, Protein Design Labs, Inc., 2375 Garcia Ave., Mountain View, CA 94043. E-mail address: Back

2 Abbreviations used in this paper: CDR, complementarity-determining region; AUC0-tlast, area under the concentration-time curve; HRP, horseradish peroxidase. Back

3 M. Cole and J. Tso. Submitted for publication. Back

Received for publication June 5, 1997. Accepted for publication September 26, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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