Cancer Immunol Immunother (1988) 27:241-245 ancer mmunolggy mmunotherapy

© Springer-Verlag 1988

In vivo antitumor activity demonstrated with squamous carcinoma reactive monoclonal antibody-Vinca immunoconjugates

David A. Johnson 1'3, John L. Zimmermann I, Bennet C. Laguzza l, and John N. Eble 2

Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, In 46285, USA 2 Indiana University, School of Medicine, Veterans Administration, Richard C. Roudebush Medical Center, Indianapolis, IN 46223, USA 3 Address inquiries to: David A. Johnson, Ph.D., Connective Tissue and Monoclonal Antibody Research (MC620), Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, IN 46285, USA

Summary. An immunoconjugate (PF1/D-DAVLBHYD), made with the squamous carcinoma reactive mono- clonal antibody PF1/D and a derivative of vinblastine, DAVLBHYD, was shown to suppress established T222 hu- man tumor nude mouse xenografts using a multidose pro- tocol. Treatments of xenograft-bearing mice with free drug, free antibody, or a mixture of the two, were unsuc- cessful at achieving suppression without associated toxici- ty, using otherwise identical protocols. A Vinca conjugate with a related squamous carcinoma reactive rnonoclonal antibody, PF1/B, was shown to have similar tumor sup- pressive activity. In a dual immunoconjugate therapy pro- tocol, PF1/D-DAVLBHYD and PF1/B-DAVLBHYD had additive antitumor effects which were consistent with their complementary tumor reactivity.

Introduction

Using monoclonal antibodies to direct oncolytic drugs to tumors is a therapeutic rational with good potential. Per- haps its most significant advantage is the achievement of increased concentrations of oncolytic drug within the tu- mor mass while limiting toxic side effects commonly asso- ciated with these drugs [7, 10]. Clearly, identification and preclinical testing of immunoconjugates will require signi- ficant effort. It will be necessary to select antibodies with desirable specifidties, and drugs which retain appropriate potency when subjected to conjugation chemistries.

Several drug immunoconjugates have already been shown to have promise, including some made with deriva- tives of the Vinca alkaloids. For example, Rowland et al. reported that a melanoma-reactive antibody (96.5), an osteosarcoma-reactive antibody (791T/36), and a carcino- embryonic antigen-reactive antibody (11.285.14) were each effective at suppressing nude mouse xenograft growth as conjugates with vindesine hemisuccinate [11]. Bumol et al. reported xenograft suppressive activity with a similar con- jugate to the adenocarcinoma-reactive antibody KS1/4 [3].

We recently reported [8] that another Vinca derivative, DAVLBHYD, when conjugated to the squamous carcino-

Abbreviations: PBS, 0.01 Msodium phosphate, pH 7.4 plus 0.15 M NaC1; DAVLBHYD, 4-desacetylvinblastine-3-carboxhydrazide; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum Offprint requests to: D. Johnson

ma-reactive antibody of Fernsten et al. [5], resulted in an immunoconjugate with potent antitumor xenograft activi- ty. This conjugate, designated PF1/D-DAVLBHYD, re- tained excellent antigen binding capacity and was able to suppress the growth of short-term established tumors. We now extend that observation and demonstrate that using a different dosing protocol, PF1/D-DAVLBHYD is able to suppress established nude mouse xenografts. An addition- al squamous carcinoma-reactive immunoconjugate, PF1/B-DAVLBHYD, is also described. This conjugate has antitumor activity either alone, or in combination with PFI /D-DAVLBHYD.

Materials and methods

Monoclonal antibody. The hybridoma cell lines PF1/D, and PF1/B were generated with the P3X63Ag8 myeloma fusion partner and both secreted active antibodies of IgG3 isotype [5]. The lines were grown as ascites in pristane- primed BALB/C mice and the secreted antibody was af- finity purified on a protein A Sepharose column (Pharma- cia, Uppsala Sweden). The wash buffer was 0.01 Msodium phosphate pH 8.0, and elution was carried out with a step gradient wih 0.1 M sodium phosphate buffer pH 3.5. Elut- ed fractions were immediately neutralized with 1 M Triz- ma buffer (Sigma, ST. Louis Mo.) pH 7.4, and dialyzed against PBS. Antibody preparations were sterilized by pas- sage through 0.2 ~tm filters and stored at 4 ° C until used.

The Pl.17 hybridoma, secreting an IgG2a protein of unknown specificity, was obtained from the American Type Culture Center (Rockville, Md.). The IgG2a was pu- rified from ascites as described before. Other control an- tibodies included HLA-reactive W6/32 (Accurate Scientif- ic, Westbury, NY), and FLOPC 21 IgG3 myeloma protein (Bionetics Laboratory Products, Kensington, Md.).

Immunoperoxidase reactivity. Tumor reactivity studies were carried out using a modification [2] of the avidin-bi- otin technique. Frozen sections of tissues were cut at 5 to 6 ~tm and picked up on poly-L-lysine-coated slides, al- lowed to air dry, and acetone fixed. Following preincuba- tion with horse serum, sections were sequentially incubat- ed with antibody, followed by biotinylated horse anti- mouse Ig (Vectorlabs, Burlingame, Calif.), and a complex of avidin and horseradish peroxidase with washes between steps in PBS pH 7.2-7.4. Color was developed uisng 3,3'

242

diaminobenzidine and sections were counterstained with hematoxylin.

Drug conjugation. The DAVLBHYD [4] was generously provided by Mr. G. J. Cullinan (Lilly Research Laborato- ries), and antibodies were conjugated to DAVLBHYD us- ing techniques detailed elsewhere [8]. Briefly, antibody was concentrated to approximately 10 mg/ml by vacuum dial- ysis in PBS and subsequently dialyzed against 0.1 M sodi- um acetate buffer pH 5.6. Treatment of this solution near 0°C with 160 mM sodium metaperiodate resulted in oxi- dized antibody which was purified by Sephadex-G25 chro- matography in 0.1 M sodium acetate buffer pH 5.6. This material was coupled with DAVLBHYD (5 mM, 4 ° C, 24 h) and purified by Sephadex G-25 chromatography in PBS, pH 7.4.

Note that antibody-drug conjugate concentrations and doses indicated were based on the drug (Vinca) content of the conjugate. Drug content of the conjugates was deter- mined using spectrophotometric methods as described elsewhere [8].

Tissue culture. The human cell line T222 (lung squamous carcinoma) [9] was maintained in DMEM plus 10% FCS plus 50 txg/ml gentamicin and adapted to roller bottles for use in nude mouse xenograft studies.

Membrane fluorescence. Target cells were suspended in trypsin/EDTA (Gibco, Grand Island, NY) and incubated with antibody diluted in DMEM plus 10% FCS on ice for 45 min. Preliminary observations indicated that the L/1C2 antigen was trypsin stable (unpublished observation). They were then washed twice with medium and incubated for an additional 45 rain with fluorescein-labeled goat F(ab')2 an- ti-mouse IgG (Tago, Burlingame, Calif.) at 20 ~g/ml medi- um. After additional washes, the cells were either ex- amined immediately by UV microscopy or fixed with 1% formalin in PBS and examined with an EPICS/Coulter Mark IV cell analyzer.

Chromatography. Aggregate formation was evaluated us- ing a Superose 12 HR10/30 gel filtration column (Pharma- cia) and a Pharmacia FPLC system [1]. Samples were ap- plied in 0.3 ml 0.1 M Trizma buffer pH 8.0 plus 0.1 M NaCI and eluted in the same buffer at 0.5 ml/min.

Nude mouse xenografts. Outbred nude mice, obtained from Charles River (Boston, Mass.), were maintained in sterile laminar air flow facilities and provided with sterile water and sterile chow ad libitum. Mice approximately 2 months of age were inoculated s.c. in the right flank with 1 x l 0 7 T222 tumor cells in 0.2 ml sterile PBS 24 h after re- ceiving 350 R gamma irradiation. The animals were treat- ed by i.v. injection in the tail vein at the indicated time points after tumor implantation. Tumor measurements were taken in two dimensions and mass was estimated us- ing the formula [6]: [(length) (width2)/2]. Where indicated, inhibition of growth was calculated relative to growth in control animals injected with PBS diluent alone. Control groups contained 10 mice, with the test groups containing 5 mice each. The student t-test was used to determine the significance of differences in mean tumor masses, and SEs are indicated were appropriate.

Results

lmmunoperoxidase reactivity

The reactivities of PF1/D and PF1/B were evaluated on serial sections from five different human squamous carci- nomas as summarized in Table 1. Both antibodies showed some level of reactivity with each of the five tumors, but the intensity varied. The observed variation in reactivity was complementary, in that a weaker reactivity by one of the antibodies corresponded to a stronger reactivity by the other. None of the tumors showed weak reactivity with both antibodies.

Immunoreactivity and immunochemistry

Fluorescent cell analyzer profiles (Fig. 1, panel A) indicat- ed that the PF1/D and PF1/B antibodies showed similar reactivity profiles with the T222 target squamous carcino- ma cell line. FLOPC 21 IgG3 myeloma protein and diluent controls showed no reactivity. A positive control, W6/32, was strongly reactive.

Conjugation of PF1/D with DAVLBHYD yielded a conjugate with approximately 5 moles of drug/mole of an- tibody, as was observed previously with this antibody [8]. The PF1/B antibody gave similar results, with approxi- mately 5 moles of drug conjugated/mole of antibody. Fig- ure 1, panel B shows that the immunoreactivity of the PF1/B antibody was only slightly reduced after being con- jugated to DAVLBHYD (85% antigen binding capacity retained). A similar result was observed with the PF1/D-DAVLBHYD conjugate was reported else- where [8].

Superose 12 chromatography of PFI/B-DAVLBHYD revealed the presence of approximately 10% aggregate (not shown) a result similar to that observed previously with the PF1/D-DAVLBHYD [8].

Control nontumor-binding IgG2a was conjugated to DAVLBHYD with similar results, yielding a conjugate

Table 1. Human squamous cell carcinoma reactivity a

Site Diagnosis PF1/D PF1/B reactivity reactivity

1. Larynx Squamous cell + + + + carcinoma Well differentiated

2. Hypopharynx Squamous cell + + + + sinus piriformis carcinoma

Moderately differ- entiated

3. Larynx Squamous cell + / - + + carcinoma Well differentiated

4. Pulmonary Squamous cell + + + metastasis from carcinoma sinus piriformis primary

5. Lung Squamous cell + + + + + carcinoma

a Score: + + +, Intense staining on all ceils; + +, Moderate staining on most cells; +, Weak overall staining or moderate staining on < 50% of cells; + / - , Faint staining overall or moder- ate staining on only a few cells; -, Negative

A B

PF,,D81 B ] i i

"1 I 11 . . . . i i . . . . . , .

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D i l u e n t 34

Log Fluorescence Fig. l . Fluorescent cell analyzer profiles. Panels labeled PF1/D and PF1/B depict reactivity of unmodified antibody at 5 p.g/ml per 1 x 106 T222 target cells. Panel B depicts the reactivity of the PF1/B antibody prior to and after conjugation to DAVLBHYD (conjugation ratio 5). Nonspecific binding to target cells was con- trolled for with a myeloma FLOPC21 IgG3. W6/32 served as a positive control. For each determination, 10,000 cells were ana- lyzed. Mean channel fluorescence is indicated

with a conjugat ion ratio of approximate ly 5 with minimal aggregate content.

PF1/D-DA VLBHYD-indueed tumor suppression

Nude mice had 1 x 107 T222 human squamous carc inoma cells implanted on day 0, and 7 days later the tumors were approximate ly 100-200 mg as est imated from cal iper mea- surements, with all animals in the exper iment bearing tu- mors of similar size. The mice were injected i.v. on days 7, 9, 11, and 13 with P F 1 / D - D A V L B H Y D , Ig-control- DAVLBHYD, free D A V L B H Y D , or a mix of free P F 1 / D plus free DAVLBHYD. Conjugate and free Vinea doses were based on Vinea content , with the animals in this ex- per iment being given 2 mg (Vinea)/kg per injection. The free ant ibody control was adminis tered at a dose equiva- lent to the protein content of the conjugate prepara t ions (75 mg (pro te in) /kg per injection). Tumor measurements were made at the t ime points indica ted on Fig. 2.

As shown in Fig. 2 (upper panel), only animals treated with P F 1 / D - D A V L B H Y D showed clear reduct ion in the size of tumors. By day 13 1 of 5 animals in that group was tumor free, and regression cont inued until day 17 when 2 of 5 animals were tumor free. Both of these animals bore tumors of approximate ly 100 mg at the start of t reatment on day 7. The tumors on the remaining 3 mice averaged 8 2 + 11 on day 17, approx imate ly the same size they had been on day 7 at which poin t the group had averaged 99 + 13 rag. By the day 26 measurement , the average tumor

243

7-DAY ESTABLISHED T222 SQUAMOUS CARCINOMA XENOGRAFTS

MICE DOSED* LV. DAYS 7, 9, 11, 13

VINCA: 2 MG/KG PROTEIN: 75 MG/KG

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O~ : : ~ ; ~ : * ; ¢ ; I I l I I I I 2 88ool [

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Fig. 2. Regression of established xenografts with PFI/D- DAVLBHYD. Significance relative to the diluent-treated controls was determined using the Student t-test with these results: PF1/D-DAVLBHYD-treated mice, day 13 P <0.05; day 17 P <0.01; day 26 P <0.001. Free DAVLBHYD-treate~! mice, day 11 P <0.01; day 13 P <0.002. Free PF1/D + DAVLt~HYD, day 11 P <0.01. Other groups and time points were not signifi- cantly different from diluent controls (P > 0.05)

size in this group had increased, though 1 mouse remained tumor free throughout this period. The exper iment was terminated at this point.

The IgG2a-con t ro l -DAVLBHYD failed to regress tu- mors, with all animals having larger tumors at the end of the experiment than they did pr ior to treatment. Tumor progression appeared to be slightly inhibited in this group relative to the di luent control group, but the differences were not statistically significant.

Figure 2 (lower panel) shows groups from the same ex- per iment which were treated with free D A V L B H Y D or a mix of free D A V L B H Y D plus free P F 1 / D . Both prepara- tions resulted in initial tumor responses, but resulted in toxicity (100% lethality) pr ior to complet ion of the ex- periment.

PFI/D-DA VLBHYD and PF1/B-DA VLBHYD dual immunoconjugate tumor suppression

T222 squamous carc inoma tumors were established on the flanks of nude mice exactly as descr ibed before. Palpable tumors were present on all mice at day 3. On days 3, 6, and 9 the mice were injected i.v. with conjugates as indicated in Fig. 3 and the tumors present on the animals were mea- sured on day 22. At that t ime, 4/5 mice treated with P F 1 / D - D A V L B H Y D at 2 mg (Vinea)/kg were tumor free ( > 90% tumor suppression). Significant tumor suppression ( > 50%) was observed in the group treated with this conju- gate at 1 mg (Vinea)/kg.

244

z 0

_T: Z F- Z Ld L3 rY i , i 13-

3-DAY ESTABLISHED T222 SQUAMOUS CARCINOMA XENOGRA~S DOSED I.V. DAYS 3, 6, 9 AND MEASURED DAY 22

(*TUMOR FREE/TOTAL) 1 0 0

90

80

70

60

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(.,9 t,9

836 MG

PF1/D- PF1/B- 50/50 DAVLBHYD DAVLBHYD MIX

1 2 MG/KG k'x'~ 1 MG/KG r%-70.5 MG/KG

Fig. 3. Individual and mixed conjugate tumor suppression. The 3-day established T222 xenografts were treated as indicated. Per- cent inhibition was determined relative to a diluent-treated con- trol group. The size of the tumors in the control group is indicated on the right axis. Significance relative to the diluent treated con- trois was determined using the Student t-test with these results: PF1/D-DAVLBHYD: 2mg/kg P <0.001; 1 mg/kg P <0.02; 0.5mg/kg P >0.05. PF1/B-DAVLBHYD: 2mg/kg P <0.001; 1 mg/kg P <0.05; 0.5mg/kg P >0.05. 50/50 mix: 2mg/kg P <0.001; 1 mg/kg P <0.05; 0.5 mg/kg P >0.05

A similar profile was seen with the PF1/B- DAVLBHYD conjugate, with 5 of 5 mice being tumor free in the 2 mg (Vinca)/kg group and > 30% suppression ob- served in the 1 mg ( Vinca)/kg group.

Additional animals were treated with a mixture of PFI /D-DAVLBHYD and PF1/B-DAVLBHYD. The indi- cated doses represented the total amount of conjugate in- jected. Overall, the results showed a profile similar to that seen wih either conjugate alone, with 5 of 5 animals in the 2 mg (Vinca)/kg group being tumor free (100% tumor sup- pression). The 1 mg (Vinca)/kg group showed > 50% sup- pression, not significantly different from that observed with similar concentrations of either conjugate alone, though 2 of 5 mice in that group were tumor free.

Discussion

Using a 3-day established T222 human squamous carcino- ma xenograft model, we showed previously [8] that the an- tibody- Vinca conjugate, PF1/D-DAVLBHYD, is superior at suppressing the growth of human tumor xenografts when compared to irrelevant conjugates, free antibody, or free drug. An important question was whether PF1/D-DAVLBHYD would be able to suppress more well-established tumors. In the present studies, T222 tu- mors were grown on the flanks of nude mice for 7 days prior to treatment, resulting in tumors of approximately 100-200 mg in size. The mice were injected in the tail vein with conjugates and controls on days 7, 9, 11, and 13. As shown in Fig. 2, PF1/D-DAVLBHYD had good antitumor activity, with a growth plateau in 3 animals and 2 animals rendered tumor free 4 days after the last treatment. How- ever, 1 apparently tumor free animal rebounded, with a tu- mor appearing by the time of the final measurement on day 26. The reason for this intragroup heterogeneity is un- clear, as it might have been predicted that all tumors

would uniformly be reduced in size. It is possible to specu- late, though, that differences in intatumor vascularity may have limited access of the conjugate to all areas of any giv- en tumor, resulting in a more limited response.

In agreement with our earlier findings [8], a nontumor- binding antibody-DAVLBHYD conjugate had no signifi- cant tumor suppressive activity. Although this IgG2a con- trol differed in isotype from the active IgG3 antibody, it served well as a control, having similar drug and aggregate content. Available IgG3 myeloma controls (including FLOPC 21), failed to meet these important criteria when conjugated to DAVLBHYD [8]. Note that nontumor-bind- ing IgG conjugates were intended to differentiate nonspe- cifically altered drug pharmacokinetic effects from anti- gen-mediated effects. The results of these studies strongly support the latter.

Free DAVLBHYD and a mix of free PF1/D plus free DAVLBHYD showed initial antitumor effects, though they were associated with toxicity with all animals in both groups dying by day 11 or 13. Similar toxicities have not been observed with the conjugated DAVLBHYD or with free PF1/D antibody [8]. Overall, this experiment allowed the conclusion that PF1/D-DAVLBHYD can be safely ad- ministered in these mouse models and can effect regres- sion of established tumors. Extended dosing protocols may be necessary, however, to accomplish complete re- gression of all treated animals.

PF1/D and PF1/B are related antibodies, reported by Fernsten et al. [5] to be reactive with similar, though not identical, squamous carcinoma-associated antigens. They may react with different epitopes on the same molecule. One presumed requirement of monoclonal antibody-based therapy is that the antibody show reactivity with a large portion of the target tumor population. Our initial analysis of PF1/D, in agreement with Fernsten et al. [5], indicated good reactivity with many, but not all squamous carcino- mas. We therefore wished to consider whether the related antibody, PF1/B, would show identical or complementary reactivity with squamous carcinoma. If the latter proved true, we wished to consider the two antibodies as partners in a dual immunoconjugate therapy protocol.

Serial frozen sections were cut from human squamous carcinoma specimens and reacted with PF1/D and PF1/B. As summarized in Table 1, the individual antibodies showed the expected heterogeneous reactivity within this group of tumors. Significantly, though, their reactivities were complementary, with all tumors being reactive with at least one of the antibodies. This led to the conclusion that the two antibodies reacted with either two similar, but distinct molecules, or with different epitopes on the same molecule. A combination or dual immunotherapy protocol with immunoconjugates of these two antibodies warranted further consideration.

The first step was to confirm the feasibility of conjugat- ing PF1/B to DAVLBHYD. The antibody behaved in a manner very similar to PF1/D, resulting in conjugation ra- tios of approximately 5 moles of drug/mole of antibody. As indicated in Fig. 1, good immunoreactivity with a tar- get squamous carcinoma cell line was retained. Superose 12 chromatography indicated an aggregate content of ap- proximately 10%, a level comparable to that observed with PF1/D conjugates [8].

A side-by-side comparison of PF1/D-DAVLBHYD and PF1/B-DAVLBHYD was made using 3-day estab-

245

lished T222 tumor xenografts. It has previously been dem- onstrated that i rrelevant conjugates and free Vinca have little ant i tumor activity in this model [81. Figure 3 shows that the two conjugates tested here had similar tumor sup- pressive activity. P F 1 / D - D A V L B H Y D at 2 mg (Vinca)/kg induced > 90% tumor suppression with 4 of 5 mice being tumor free by day 22. P F 1 / B - D A V L B H Y D at the same dose resulted in 100% tumor suppression with 5 of 5 mice being tumor free. With both conjugates, the ant i tumor ac- tivity decl ined in a dose-dependent fashion, with no signi- f icant differences between the two conjugates.

The abil i ty of a mixture of P F 1 / D - D A V L B H Y D and P F 1 / B - D A V L B H Y D to suppress tumor growth was tested. The results showed tumor suppressive activity very similar to that seen with either conjugate alone, with 5 of 5 ani- mals being tumor free at a total dose of 2 mg (Vinca)/kg. In addit ion, animals were tumor free at the lower total dose of 1 mg (Vinea)/kg, in contrast to what was observed with either conjugate alone. Although this is suggestive of synergistic activity, the mean tumor masses of all groups t reated at this dose level were not significantly different, al lowing only the conclusion that the conjugates had addi- tive effects. In either case, this result is encouraging, as it may have been found that one ant ibody had inhibi tory ac- tivity for the other, perhaps due to steric h indrance by b inding to closely si tuated epitopes.

In summary, these studies have shown that the squa- mous carcinoma-react ive Vinea alkaloid conjugate P F I / D - D A V L B H Y D can suppress established human tu- mor nude mouse xenografts. In addi t ion, the conjugat ion of D A V L B H Y D to another squamous carc inoma reactive ant ibody (PF1/B) was accompl ished successfully and this conjugate showed good ant i tumor activity. When the P F 1 / D - D A V L B H Y D and P F 1 / B - D A V L B H Y D conju- gates were used in combinat ion , addit ive xenograft sup- pression was observed. This result, combined with the ob- served addit ive squamous carc inoma reactivity profile of these two ant ibodies, makes them reasonable candi- dates for addi t ional deve lopment as therapeut ic agents. Future efforts will be directed toward evaluating potent ia l toxicities due to the normal tissue reactivities of these antibodies.

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Received February 3, 1988/Accepted May 26, 1988