Influence of Affinity and Antigen Internalization on the Uptake and Penetration of Anti-HER2 Antibodies in Solid Tumors

Antibodies Cell Lines and Radiolabeling

C6.5 IgG1 affinity variants were produced and characterized exactly as described previously (12). Trastuzumab (Herceptin) was obtained through the Fox Chase Cancer Center's pharmacy and diluted to 1mg/mL prior to use. Prior to radiolabeling of the C6.5 variants, storage buffer was exchanged to sterile PBS using 4mL 10000 MWCO Amicon centrifugal filters (Millipore). No buffer exchange was performed on trastuzumab. For radioiodination reactions, 1μCi ¹²⁵I-Na (Perkin-Elmer) per microgram of IgG to be labeled was dissolved in 50μL 0.2M phosphate buffer (pH 7.6), applied to Pierce Pre-Coated Iodination Tubes (Thermo Scientific), and mixed at room temperature for 5min. The IgG was added to the iodination tube and mixed for 3min at RT. The labeling reaction was terminated by removal of protein from iodination tube followed by purification over a Sephadex G-50 spin column (Sigma) pre-equilibrated with PBS (pH 7.4). The SK-OV-3 cell line (ATCC HTB-77) was obtained from the ATCC and was passaged for fewer than 6 months. The ATCC has verified the identity of this cell line by methods including short tandem repeat (STR) profiling.

Prior to radiometal chelation, trace metal contaminants were removed from all buffers and water by passage over a 2.5×50cm column (Kontes) with Chelex 100 resin (Bio-Rad). All IgGs were exchanged into 500mM NaCl, 50mM carbonate buffer (pH 8.5) and conjugated with a 50 fold molar excess of CHXA-A′′-DTPA (Macrocyclics) for 18hr at RT in the dark. Excess chelator was removed by buffer exchange, four times, into 150mM acetate buffer (pH 5.3). With specific activity diluted to 20μCi/μL in 50mM HCl, ¹¹¹InCl₃ (Perkin-Elmer) was added to 500mM acetate buffer to yield ¹¹¹InOAc in 150mM buffer. At a ratio of 1.2μCi/μg, IgG-DTPA conjugate was mixed with ¹¹¹InOAc for 18hr at RT in the dark. Excess ¹¹¹In was chelated with addition of 100mM EDTA (final concentration to 5mM to 10mM) and separated over Sephadex column as above.

Quality control of the radiolabeled antibodies was performed as previously described (8). Briefly, purity of all radiolabeled proteins was assessed using TEC-Control Chromatography Strips (Biodex). Immunoreactivity (IR) after labeling was verified by assaying 10ng labeled IgG against 3×10⁶ SK-OV-3 cells and determining percent bound to cells and supernatant. The average %IR after radioiodination reactions from multiple studies was 52.9, 77.4, 57.3, 65.7, and 87.9% for G98A, C6.5, ML39, H3B1, and trastuzumab respectively. The average %IR after radioindium chelation from multiple studies was 56.1, 71.2, 62.3, 70.3, and 62.2% for G98A, C6.5, ML39, H3B1, and trastuzumab respectively.

Internalization Assays

SK-OV-3 cells were harvested in the logarithmic phase of growth and incubated with the ¹²⁵I labeled IgGs at 450 ng per 5 × 10⁵ cells on ice for 1 hour. Cells were washed twice with cold PBS containing 1% BSA, aliquoted to 18 tubes, and incubated at 37°C, 5% CO₂. After 0, 1, 2, 4, 8, 24hr, the cultures were separated into catabolized, dissociated, cell surface bound and internalized fractions as described below. Cell suspensions were pelleted in a refrigerated centrifuge at 1500 rpm for 5 min. Supernatants were transferred to Microcon YM-30 centrifugal filter unit (Millipore), followed by centrifuge at 14000 rpm for 20 min. The supernatants, which passed through the filters, represented the catabolized fractions, while what remained in the filters comprised the dissociated fractions. Cell surface bound IgGs were eluted from cell pellets with freshly made stripping buffer (2M Urea, 50 mM Glycine, 150 mM NaCl, pH 2.4). The elution step was repeated three times to remove over 98% of cell surface antibodies. The cell pellets after elution contained the internalized fractions. All the fractions were assayed in a Packard E5002 gamma counter (Perkin-Elmer).

Biodistribution Studies

Antibody biodistribution studies were conducted as previously described (13). Four to six week old female scid mice were obtained from the Fox Chase Cancer Center animal facility. Tumors were implanted by s.c. injection of 2.5×10⁶ SK-OV-3 cells into the abdomen of the mice. After 6–8 weeks, the tumors reached a size of ≥ 100mg. Mice to be used in ¹²⁵I experiments were given Lugol's solution in their drinking water to prevent thyroid accumulation of radioiodine. The pool of unoccupied Fc receptors in scid mice was blocked by i.p. administration of 1mg murine IgG2a-κ (Sigma) in order to prevent altered pharmacokinetics. Biodistribution studies were initiated 48hr later with the i.v. injection of 20μg radiolabeled antibody. Within 5–10min, blood samples were taken and whole body counts were measured on a Captus 600 or Captus 3000 multichannel analyzer (Capintec) in order to determine the total injected dose. For analysis of the full C6.5 IgG affinity series, tumor, tissue and blood distribution was assessed at 24hr and 120hr. For C6.5 and trastuzumab time course experiments, these measurements were taken at 8, 24, 48, 72, and 120hr after injection. Cohorts of 5 mice were employed at each time point, and blood samples were taken immediately before euthanasia. Immediately after euthanasia, whole body counts were measured and tumors and normal organs were removed and weighed. The percent injected dose per gram (%ID/g) was determined as previously described (14) for each organ and blood sample after determining the cpm retained in the samples using a Packard E5002 gamma counter (Perkin-Elmer). All measurements were corrected for radioactive decay. Representative results from multiple studies are shown. Statistical significance was determined using an unpaired t test using GraphPad software.

Immunohistochemistry Penetration Studies

With the exception of Lugol's solution, mice and tumors were handled as described above. Antibodies were obtained and buffer exchanged to sterile PBS as described above. Consistent with previous models of tumor penetration (8), 500μg of each antibody was injected i.p. into cohorts of three mice 48hr after administration of the blocking IgG2a-κ. 120hr later, mice were euthanized and tumors removed and fixed in 10% formalin for 24–48hr. After embedding in paraffin blocks, sections were cut and mounted on slides for IHC. After depariffination and rehydration using standard methodologies, primary antibodies were applied. Human HER2 was detected using rabbit anti-human c-erbB-2 oncoprotein polyclonal antibody (A0485, Dako). Blood vessels were detected using rat antimouse CD31 (553370, BD Pharmigen) monoclonal antibody. The i.p. administered antibodies were detected in tumor sections with biotinylated goat anti-human IgG antibodies (Vector Labs). For double staining of CD31 and human IgG, the sections were treated with 0.06% Pronase E (Sigma) in 0.5M Tris buffer, pH 7.5 for 10 min at room temperature and then incubated with 3% H₂O₂ for 10 min to block endogenous peroxidase activity. Sections were blocked with normal rabbit serum. Tumor sections were incubated with rat anti-mouse CD31 (PECAM-1) monoclonal antibody at a 1:300 dilution overnight at 4°C. Negative controls were incubated with the rat serum IgG at the same protein concentration. All sections were washed in PBS containing 0.05% Tween-20, and then incubated with goat anti-rat IgG (BD, Pharmingen) at a 1:200 dilution for 30 min at room temperature. After washing, the sections were incubated in an avidin peroxidase reagent (SA-HRP 1:100 dilution Perkin Elmer) for 30 min. After washing, a Biotinyl-Tyramide enhancement kit (TSA™ Biotin Tyramide Reagent Pack, Perkin Elmer) was used according to the manufacturer's instructions. After washing, the sections were incubated in peroxidase substrate (DAB, Sigma) for 5 min. Next, we used biotinylated goat anti-human IgG, dilution 1:4000 overnight at 4°C followed by streptavidin peroxidase reagent (Bio Genix) that was developed with the Vector VIP substrate for peroxidase as per manufacturer's instructions (Vector Labs). This produces a purple immunostaining that contrasted with the brown stain of CD31 blood vessels. The average tumor penetration of the MAbs was quantified using de-identified samples by measuring the distance of anti-human IgG staining from blood vessels. For each MAb, four isolated blood vessels were randomly selected per slide on three different slides (two for ML39). The distance in microns the MAbs penetrated was measured twice for each vessel with each measurement taken on opposite sides, resulting in 8 measurements per slide. Statistical significance was determined using an unpaired t test using GraphPad software.

High affinity IgG exhibits less tumor retention and accumulation than moderate affinity clones in vivo

In order to probe the relationship between IgG binding affinity and tumor retention, a panel of anti-HER2 IgG's derived from a series of C6.5 scFv affinity variants (9, 10) was utilized (Supplemental Table 1) At the amino acid sequence level, the series of IgGs derived from the C6.5 clone are identical except for point mutations in the CDR affecting affinity to HER2. Like their parental scFv clones, these IgGs all target the same epitope on HER2 with distinct logarithmic increases in monovalent binding affinity from 270nM to 0.56nM. With the exception of the lowest affinity IgG, only minor differences in the functional (bivalent) affinities of the IgGs were observed (12). This was likely due to the limitations of the assays available to accurately measure very high multivalent affinities. For the reason of clarity, our observations will be discussed predominantly in consideration of IgG monovalent binding affinity, however it should be noted that bivalent affinity is likely playing a significant role in our observations.

To determine tumor uptake, 20μg radioiodinated (¹²⁵I) anti-HER2 IgGs were injected intravenously into scid mice with established subcutaneous SK-OV-3 human ovarian carcinoma tumor xenografts overexpressing HER2. For each MAb (G98A [270 nM], C6.5 [23 nM], ML39 [7.3 nM], and H3B1 [0.56 nM]) cohorts of five mice were euthanized after 24hr and 120hr, and the amount of radiolabeled antibody remaining in tumors, normal tissue, and circulation was determined. Multiple studies were conducted, and the results of a representative experiment are shown in Table 1 and Figure 1. While no consistent difference in accumulation in normal tissue or rates of systemic clearance was observed for the anti-HER2 affinity series (Table 1), it was repeatedly observed that the high affinity IgG H3B1 (0.56nM monovalent KD) had the significantly lower tumor accumulation at both 24hr (p=0.0476) and 120hr (p<0.0001) (Figure. 1, dark gray bars) compared with C6.5 IgG (Figure. 1, light gray bars). The low affinity and moderate affinity IgGs G98A and ML39 (270nM and 7.3nM monovalent KD respectively) had slightly lower accumulation in the tumor than C6.5, although these differences were not statistically significant (Figure. 1, black and white bars).

IgG binding affinity limits penetration into tumors in vivo

While the biodistribution study described above revealed varying tumor retention of antibodies based on antibody intrinsic affinity, it cannot address antibody localization within the tumors. Therefore to investigate the relationship between the distance an antibody penetrates into the tumor from a blood vessel and affinity, we administered a 500μg intraperitoneal dose of the each unlabeled affinity variant and trastuzumab to scid mice bearing established SK-OV-3 tumors. After 120hr, tumors were harvested and stained for human HER2, tumor vasculature (CD31), and human IgG to determine antibody penetration as a function of affinity (Figure. 2). IHC revealed no significant differences in tumor structure (Figure. 2A) or in the relative expression of hHER2 on the surface of the tumor cells (Figure. 2B). Dual staining (Figure. 2C–H) for the blood vessels (CD31, brown) and for human IgG (purple) revealed significant differences in the penetration of the antibodies from blood vessels into the tumor with an apparent threshold occurring between 23nM and 7.3nM monovalent affinity. G98A, the MAb with the lowest monovalent affinity (270nM), exhibited the greatest average penetration distance from the blood vessels (84.8 microns). C6.5, the MAb with a moderate monovalent affinity (23nM), penetrated to an average distance of 59.7 microns. Further increases in monovalent affinity (e.g., ML39, 7.3nM; H3B1, 0.56nM; and trastuzumab, 0.09nM) were associated with significantly decreased tumor penetration (23.3, 20.4 and 37.2 microns, respectively) (p<0.0001 for C6.5 IgG vs all other antibodies). This observed degree of tumor penetration is within the predicted range modeled by others (based upon published data sets from a number of groups) of approximately 20 and 130 microns when a 500μg dose is governed by antigen turnover and systemic clearance respectively (5).

Affinity and the rate of antigen internalization dictate the sub-cellular distribution of anti-HER2 IgGs

Antigen-mediated internalization of antibodies has been shown to lead to their degradation in a variety of models in vitro (4, 15, 16) and in vivo (17). Furthermore, HER2 is a dynamic antigen whose internalization varies with receptor states such as hetero/homo-dimerization, and ligand binding (18), but is not affected by binding of trastuzumab (19). Therefore, in order to investigate the impact of antibody affinity on sub-cellular distribution, an internalization assay was conducted with ¹²⁵I labeled IgGs as previously described (19). For the C6.5 variants and trastuzumab, 450ng ¹²⁵I labeled IgG was incubated with 5×10⁵ SK-OV-3 cells and the degree of catabolism, dissociation, retention on the cell surface, and retention inside of cells was determined after 0, 1, 2, 4, 8, and 24hr.

With the exception of the lowest affinity antibody, G98A, 60% to 70% of each IgG remained on the cell surface within the first four hours of the assay (Figure. 3A). After 24hrs, however, more C6.5 and ML39 remained on the surface of the cells than was observed with the highest affinity variants, H3B1 and B1D2, and trastuzumab. More than 80% of G98A dissociated in 4hrs (Figure. 3B), explaining why so little was observed on the cell surface. In contrast, C6.5 dissociated slower than G98A and the highest affinity variants and trastuzumab (Figure. 3B) exhibited the least dissociation. Very little of any of the IgGs remained intact inside the cells despite their large differences in affinity (Figure. 3C). Due to pronounced dissociation from the cells, very little G98A was degraded (Figure. 3D). The degradation of the other tested IgGs increased over time. Since the monovalent dissociation rate constant (k_off) for the IgGs has not been measured, catabolism after 24hr was correlated with the k_off rate of the parental scFv molecules. For G98A, C6.5, and ML39 with scFv-based off rates faster than 7×10⁻⁴s⁻¹ (10), catabolism increased with affinity (decreased off rate) (Figure. 3E). For MAbs with slower rates of dissociation, H3B1, B1D2, and trastuzumab (20), catabolism and degradation was no longer impacted by further decreases to the offrate.

Reported HER2 internalization rates (ke) range from 1.6×10⁻³s⁻¹ to 1.6×10⁻⁴s⁻¹ (18), and are represented in Figure 3 by the shaded region. This suggests that the rate of HER2 internalization serves as an affinity threshold in determining the fate of these antibodies. Degradation increases with affinity as long as the koff is faster than ke, despite the ability of the antibodies to bind bivalently. Furthermore, internalization is independent of affinity and becomes totally antigen dependent when koff is slower than ke, and these interactions, as with many other antibodies, can be considered irreversible as suggested previously by others (15, 16).

Pharmacokinetic analysis reveals that tumors consume significant amounts of antibody in vivo

Of the antibodies that showed significant retention on the surface of SK-OV-3 cells, C6.5 IgG exhibited the least degradation, while trastuzumab exhibited the highest level. We hypothesized that this would lead to distinct rates of uptake and clearance stemming from the more rapid turnover of trastuzumab relative to C6.5. To address this hypothesis, two biodistribution experiments were conducted to compare the systemic and tumor pharmacokinetics of C6.5 and trastuzumab. These experiments were designed to exploit the metabolic differences between ¹²⁵I and ¹¹¹In radiolabels in order to determine the relative impacts of internalization and degradation. Once internalized, radio-iodotyrosine metabolites from ¹²⁵I-labeled MAbs are rapidly eliminated by cells, while radio-indium metabolites from ¹¹¹In-labeled MAbs residualize and accumulate in cells following their degradation (21–23). Therefore the ¹²⁵I radiolabel is representative of intact surface bound and unbound antibodies in the tumor microenvironment, and the ¹¹¹In radiolabel is indicative of both intact and the accumulated degraded antibody.

In Figure 4, the %ID/g in blood (empty symbols with gray lines) and in tumor (filled symbols with black lines) is presented for ¹²⁵I (triangles) and ¹¹¹In (circles) labeled C6.5 IgG (A) and trastuzumab (B). For both C6.5 and trastuzumab, the rate of clearance from circulation was independent of the radiolabel employed. Tumor accumulation of the radioiodinated antibodies peaked at 48hr and 24hr for C6.5 and trastuzumab, respectively. After 24hr, ¹²⁵I-trastuzumab levels in the tumor decreased at the same rate as the antibody from circulation. In contrast, ¹²⁵I-C6.5 %ID/g in the tumor persisted at the same level between 48hr and 72hr despite much lower blood concentrations. Therefore, relative to trastuzumab, ¹²⁵I-C6.5 clearance from the tumor was delayed, even following first-pass clearance of both proteins. In contrast with the ¹²⁵I tumor retention profiles, between 48hr and 120hr, both antibodies possessed much higher ¹¹¹In tumor retention. Thus, both antibodies undergo in vivo internalization and degradation. Because of the increased rate of ¹²⁵I-trastuzumab clearance relative to C6.5, and the large difference between ¹²⁵I-trastuzumab (intact) and ¹¹¹In-trastuzumab (intact and degraded) accumulation in the tumor over time, we conclude that, consistent with the in vitro data (Figure 3), trastuzumab is internalized and degraded in vivo by tumors more than C6.5

Total amount of antibody delivered to tumor in vivo is independent of affinity

Based on the observations of the in vitro internalization assays and the PK study above, the higher affinity clones should be degraded to a higher extent than lower affinity clones in vivo. To test this hypothesis, an ¹¹¹In biodistribution study was performed for the anti-HER2 affinity series (Figure. 5) to compare with the ¹²⁵I results presented in Figure 1.

For all IgGs, samples were collected 24hr and 120hr post-injection of 20 μg ¹¹¹In labeled protein. While no differences were observed in systemic clearance or normal tissue distribution for the affinity variants (Table 2), the %ID/g of all the tested antibodies in the tumor increased over time. . Accordingly, factors such as extravasation from blood vessels were apparently unaffected by affinity. For the three IgGs with monovalent KD below 10⁻⁸M, the accumulation of the ¹¹¹In-labeled forms in tumor, comprising both intact and degraded IgG, was identical, while ¹¹¹In-G98A tumor accumulation was significantly lower (P<0.05 for all three IgGs at 120hr). In contrast with these results, affinity higher than that of C6.5 IgG leads to the tumor retention of less intact radioiodinated antibody due to in vivo internalization and degradation, illustrating the magnitude of escalating MAb degradation by the tumors as affinity increases from C6.5 to H3B1.