Curing mice with large tumors by locally delivering combinations of immunomodulatory antibodies

Abstract

Purpose

Immunomodulatory mAbs can treat cancer, but cures are rare except for small tumors. Our objective was to explore whether the therapeutic window increases by combining mAbs with different modes of action and injecting them into tumors.

Experimental Design

Combinations of mAbs to CD137/PD-1/CTLA4 or CD137/PD-1/CTLA4/CD19 were administrated intratumorally to mice with syngeneic tumors (B16 and SW1 melanoma, TC1 lung carcinoma), including tumors with a mean surface of ~80mm². Survival and tumor growth were assessed. Immunological responses were evaluated using flow cytometry and qRT-PCR.

Results

Over 50% of tumor-bearing mice had complete regression and long-term survival after tumor injection with mAbs recognizing CD137/PD-1/CTLA4/CD19 with similar responses in 3 models. Intratumoral injection was more efficacious than i.p. injection to cause rejection also of untreated tumors in the same mice. The 3 mAb combination could also induce regression but was less efficacious. There were few side-effects and therapy resistant tumors were not observed. Transplanted tumor cells rapidly caused a Th2 response with increased CD19 cells. Successful therapy shifted this response to the Th1 phenotype with decreased CD19 cells and increased numbers of long term memory CD8 effector cells and T cells making IFNγ and TNFα.

Conclusion

Intratumoral injection of mAbs recognizing CD137/PD-1/CTLA4/CD19 can eradicate established tumors and reverse a Th2 response with tumor-associated CD19 cells to Th1 immunity while a combination lacking anti-CD19 is less effective. There are several human cancers where a similar approach may provide clinical benefit.

Introduction

Over a century ago, Coley reported that injecting certain bacterial toxins occasionally induced complete regression (CR) and cure of advanced cancers (1), and it has been known for decades that tumors, including those in humans, are recognized by the host’s immune system (2, 3). However, the role of immunological responses to most human cancers was questioned for many years and therapeutic tumor vaccination has not been sufficiently effective to become part of the clinical mainstay (4). A strong interest in cancer immunotherapy is now emerging as a result of encouraging clinical data (5–8). A major goal is to overcome the high immunosuppression in the tumor microenvironment (9) and the appearance of therapy resistant tumor variants as a result of immunological editing (10).

The identification of signals which regulate the immune response (11–13) played a key role to provide novel therapeutic approaches, e.g. using agonistic and antagonistic immunomodulatory mAbs to lymphocyte receptors and/or their ligands to induce a tumor-destructive Th1 response (14). MAbs to CTLA4 were the first shown to have anti-tumor activity in mouse models (15), followed by mAbs to CD137 (16), and mAbs to CD3 plus CD28 were found to induce a tumor-selective Th1 response by lymphoid cells from patients with advanced cancer when added to cultured autologous tumor cells (17). A variety of additional mAbs were soon reported to cause a tumor-destructive immune response in preclinical models, including mAbs to PD-L1 (18, 19), CD40 (20), Ox40 (21), PD-1 (22) and CD20 (23) and antibody combinations were often more efficacious than single mAbs (24). Several mAbs also yield clinical benefit, including mAbs to CTLA4 (25, 26), PD-1 (27), PD-L-1 (28) a combination of mAbs to CTLA4 plus PD-1 (29), and of mAbs to CD137 plus PD-1 (30). However, the frequency of CR and cure remains low (31).

We hypothesized that the therapeutic efficacy can be improved by combining immunomodulatory mAbs with different mechanisms of action including a mAb to CD19 to counteract tumor-promoting B cells (32) and/or a subpopulation of dendritic cells prone to induce immunological tolerance (33) and by injecting the mAbs intratumorally (i.t.) to create a local inflammation with bystander killing (34) which may prevent the emergence of therapy resistant variants. Our findings support the hypotheses by demonstrating frequent CR and long term survival in all of 3 mouse tumor models (SW1 and B16 melanoma, TC1 lung carcinoma) and show that injection of tumors is therapeutically more efficacious than systemic administration to induce a strong local and systemic response. CR and long term tumor free survival were achieved also when the tumors were large at the onset of treatment. Responses were consistently associated with a shift in the tumor microenvironment from the Th2 type to Th1 immunity with long term memory T cells and expression of genes encoding IFNγ and TNFα, and therapy resistant cells were not detected. Importantly, the level of side-effects was low.

Based on these findings and our previous demonstration that a combination of mAbs to CD137/PD-1/CTLA4 prolongs survival of mice with the ID8 ovarian carcinoma (35, 36), as well as on data from others (as cited above), we conclude that many cancers can be cured by engaging immunological mechanisms of the Th1 type and we expect that increased knowledge about the underlying mechanisms will revolutionize cancer therapy. Encouraged by clinical responses with mAb combinations we feel that the 4 mAb combination should be evaluated in humans.

Materials and Methods

Tumor Lines and Mice

SW1 is a clone from the K1735 melanoma of C3H origin with low immunogenicity and expression of MHC class I and II, B16/F10 is clone from a spontaneous C57BL/6 melanoma and TC1 is a clone from a C57BL/6 lung carcinoma that was transfected with HPV-16 E6 and E7. Cells were cultured in IMEM supplemented with 10% fetal calf serum (FBS) (Atlanta Biological, Norcross, GA), and 1% penicillin and streptomycin and suspensions prepared and transplanted to mice. Six to eight-week female C57BL/6 and C3H mice were purchased (Charles River Laboratories, Wilmington, MA). The animal facilities are certified by the Association for Assessment and Accreditation of Laboratory Animal Care, and our protocols are approved by the institution (University of Washington).

Monoclonal antibodies for immunotherapy

The following mAbs were purchased from BioXcell: anti-CD137 (LOB12.3; Cat. #BE0169), anti-PD-1 (RMP1-14; Cat. #BE0146), anti-CTLA4 (9D9; Cat. #BE0164), anti-CD19 (1D3; Cat. #BE0150) and control (2A3; Cat. #BE0089) and administered as indicated.

Animal studies

5×10⁵ SW1 cells, 5×10⁵ TC1 cells or 1×10⁵ B16 cells were transplanted subcutaneously (s.c.) on the right flank. When mice had tumors of the size referred to in Results (either 16–25 mm² or 64–100 mm² mean surface area), they were randomized into treatment groups and injected with mAbs, 0.25 mg of each indicated mAb; the mAbs were injected intratumorally (i.t.) as indicated, at weekly intervals for 3 times, followed by 3 biweekly intervals; in some experiments (see Results) we, instead, injected the mAbs intraperitoneally (i.p.). Mice were monitored daily for side effects, two perpendicular tumor diameters were measured twice/week and tumor surfaces were calculated. Survival was recorded for each mouse and overall survival (OS) was calculated as mean±standard error (M±SEM).

To investigate therapeutic effects on a second, untreated tumor in the same mice were transplanted with 5×10⁵ SW1 cells s.c. on their right and 2.5×10⁵ SW1 cells on their left flank. When the right tumors were approx. 7–8 mm and the left ones approx. 5–6 mm mean diameter, the 4 mAb combination was injected either into the right tumors while leaving the left tumors untreated or i.p. for systemic distribution; this treatment was repeated as for the other experiments with the SW1 tumor (shown by arrows in the Figs).

For in vitro studies to investigate mechanisms, mice subjected to various treatments (including controls) as indicated in Results were euthanized and tumor draining lymph nodes (TLN), spleens and tumors were harvested. The collected tissues were prepared as described below. Mice with progressively growing tumors (progressors), whose tumors had fully regressed (regressors) as well as treated mice whose tumors started to respond (responders) or continued to grow (nonresponders) were investigated.

To obtain normal cells (presumably fibroblasts) to compare the immunological effect with that of transplanted tumor cells, one female mouse, syngeneic to the tumor being studied in parallel, was euthanized after which the lungs removed, cut into ~1 mm pieces which were incubated in IMEM medium with 500μg/ml liberase at 37°C for 1 hour. The cell suspension was filtered through a 100μm filter and washed 3 times with IMDM containing 10% FBS and then cultured similarly as the tumor cells.

Flow cytometry

Single cell suspensions from spleens and lymph nodes were prepared as published (37, 38). Tumor-infiltrating lymphoid cells (TIL) were isolated from 2 or 3 pooled tumors as described(39). Single cell suspensions were washed with FACS staining buffer and incubated with mouse Fc receptor binding inhibitor for 10 minutes before staining with antibodies of CD45 (clone 30-F11), CD3 (clone 145–2C11), CD4 (clone GK1.5), CD8 (clone 53–6.7), CD19 (clone eBio1D3), CD20 (clone AISB12), CD86 (clone GL1), CD80 (clone 16-10A1), CD11b (clone M1/70), Gr-1 (clone RB6-8C5), PD1 (clone RMP1-30), CD137 (clone 17B5), PD-L1 (clone MIH5), PD-L2 (clone 122) and CD11c (clone N418) for 30 minutes. All these mAbs were bought from eBioscience, San Diego, CA. For intracellular staining of, IFNγ (clone XMG1.2 eBioscience), TNFα (clone MP6-XT22; eBioscience), CTLA4 (clone MP6-XT22; eBioscience) and Foxp3 (clone FJK-16s; eBioscience), cells were fixed, permeabilized and stained following the instruction of Cytofix/Cytoperm kit (BD Bioscience, San Jose, CA). Flow cytometry was performed using FACSCalibur (BD Biosciences) and the leukocyte population was selected by gating CD45⁺ cells. CountBright^™ Absolute Counting Beads (Life technologies, Grand Island, NY) were mixed with the cell sample and used to calculate absolute cell numbers. The data were analyzed using Flow Jo software (Tree Star, Ashland, OR).

To investigate whether there was a functional T cell response, TLN or splenocytes were cultured for 16 hours with Cell stimulation Cocktail (ebioscience, San Diego, CA) which would induce the activation of cytokines production. After which intracellular cytokines were measured by flow cytometry.

Immunohistochemistry

SW-1 bearing mice were euthanized 7 days after their tumors had been injected once with the 3 mAb injection, and the tumors were fixed in 10% paraformaldehyde. Four-micron sections of formalin-fixed paraffin-embedded tissue were cut and placed on Superfrost Plus microscope slides (Fisher Scientific, Pittsburgh, PA). The sections were deparaffinized and rehydrated through graded alcohols. Antigen retrieval was carried out with Target Retrieval Solution (DAKO Corporation; Carpinteria, CA) in a microwave for 10 minutes. Endogenous peroxidase activity was blocked with 3% H₂O₂. The slides were washed and incubated with anti-CD4 (Novus biologicals, Littleton, CO), anti-CD8 (Biorbyt, San Francisco, CA) or anti-CD19 (Biorbyt, San Francisco, CA) mAbs overnight at 4°C, after which they were washed and incubated with Anti-Rabbit IgG-Peroxidase antibody (Sigma-Aldrich, St.Louis, MO). Color development was accomplished by incubation in diaminobenzidine (DAKO Corporation; Carpinteria, CA). The slides were counterstained by hematoxylin (DAKO Corporation) and coverslipped with permanent mounting media.

Quantitative PCR

Collected TLNs, spleens and tumors (when available) were stored in RNALater (Sigma-Aldrich, St.Louis, MO) at −20°C. Total RNA was extracted from different tissues using Qiagen RNeasy Mini Kit, followed by cDNA synthesis using iScript^™ Reverse Transcription Supermix (Bio-rad, Hercules, CA). Subsequently, cDNA was used to measure the mRNA level of TNFα, IFNγ, IL4, Tbx21, Gata3, perforin and granzyme B using qRT-PCR on ABI Viia7 Real-Time PCR System (Applied Biosystems).The relative quantification was performed using the Comparative CT method described by the manufacturer.

Statistics

Results were expressed as mean ± SEM. Student’s t test was used to compare the statistical difference between two groups and one-way ANOVA was used to compare three or more groups. Kaplan-Meier survival analyses were performed using GraphPad Prism 5, and the Gehan-Breslow-Wilcoxon test was used to determine significance. p < 0.05 was considered to be statistically significant.

RESULTS

Certain combinations of immunomodulatory mAbs induce CR

We previously reported that i.t. injection of anti-CTLA4 plus anti-PD-1 plus anti-CD137 mAbs (“the 3 mAb combination”) has efficacy in the ID8 ovarian cancer and SW1 melanoma models (35), and our present study confirms this efficacy in the 3 models investigated, SW1 and B16 melanoma and TC1 lung carcinoma (Table 1) with CR in 26 of 40 (65%) mice with SW1, 3/10 (30%) mice with B16 and 4/15 (27%) of mice with TC1 tumors when the mAbs were first injected when the tumors had a surface area of ~25mm².

Table 1.

Significantly prolonged survival of mice with established tumors following administration of mAbs to CD137/PD1/CTLA4 or to CD137/PD1/CTLA4/CD19.

Tumor model	Survival (days, M ± SEM)
	Control	3 mAb i.t.	3 mAb i.p.	4 mAb i.t.	4 mAb i.p.
SW1 (25mm2, n=108)	14.5 ± 0.9 (0/38)π	107.2 ± 8.6*** (26/40)	61.3 ± 11 ## (5/15)	157.9 ± 9.2*** ## (14/15)	NT
SW1 (80mm2, n=15)	16 ± 1.3 (0/5)	50.6 ± 9.2** (0/5)	NT	111 ± 24.1** # (3/5)	NT
B16 (25mm2, n = 55)	8.6 ± 0.7 (0/20)	51.3 ± 19.4** (3/10)	NT	121.5 ± 16.5*** # (14/20)	40.6 ± 24.9& (1/5)
B16 (80mm2, n=35)	9.5 ± 0.9 (0/15)	29.5 ± 10.4* (1/10)	NT	77 ± 18.9*** # (5/10)	NT
TC1(25mm2, n = 45)	10.4 ± 0.8 (0/15)	61 ± 19.3* (4/15)	NT	94.5 ± 21.5*** # (7/15)	NT

^πindicates survival (tumor free)/number of treated mice 120 days after the first treatment.

(* P < 0.05, ** P < 0.01, *** P < 0.001, compared with Control group; ^# P < 0.05, ^## P < 0.01, compared with 3 mAb i.t. group; ^& P < 0.05, compared with 4 mAb i.t. group.)

In view of the involvement of B cells in Th2 mediated anti-tumor responses (40), as well as our demonstration of an increased number of CD19⁺ cells in TLN after tumor cell transplantation and the finding that tumor regression induced by the 3 mAb combination was associated with a decreased number of CD19⁺ cells in tumors and TLN, we hypothesized that an anti-CD19 mAb would have anti-tumor efficacy. While i.t. injection of an anti-CD19 mAb only slightly prolonged survival of SW1-bearing mice, as did mAbs to either CTLA4 or PD-1, combination of anti-CD19 with either of these two mAbs significantly prolonged their survival (Fig 1, p < 0.05) as did anti-CD19 plus anti-CTLA4 mAb in mice with B16 melanoma (Fig 1, p <0.05) where neither mAb was efficacious as a single agent. Importantly, addition of CD19 mAb to the 3 mAb combination significantly increased survival in all 3 tumor models (Table 1). Thus i.t. injection of anti-CD137/PD-1/CTLA4/CD19 (“the 4 mAb combination”) produced CR in 14/15 SW1-bearing mice (p<0.01), in 14/20 B16-bearing mice (p<0.05), and in 7/15 mice with TC1 tumors (p<0.05).

FIGURE 1.

Eradication of established tumors by mAb combinations. When s.c. tumors had ~25 mm² surface area, they were injected with indicated single mAbs or mAb combinations for 6 times as shown by arrows. (A) Survival curves for mice with s.c. SW1 melanoma (left), B16 melanoma (middle) or TC1 lung carcinoma (right). (B) Tumor growth curves for individual mice with s.c. SW1 melanoma (upper), B16 melanoma (middle) or TC1 lung carcinoma (bottom). n=5 for each group * p<0.05, **p<0.01, *** p<0.001. Similar results were obtained in at least three independent experiments summarized in Table 1.

We next explored the efficacy against larger tumors by treating mice which had s.c. melanoma of ~80 mm² surface area. As shown in Fig 2, anti-CTLA4 plus anti-PD-1 mAbs was not efficacious in mice with large SW1 or B16 tumors. The 3 mAb combination prolonged overall survival (OS) of SW1-bearing mice to 50.6 ± 9.2 days from 16 ± 1.3 days in controls (p<0.01) and of B16-bearing mice to 29.5 ± 10.4 days from 9.5 ± 0.9 days in controls (p<0.05), but it only induced one CR. In contrast, the 4 mAb combination induced long-lasting CR in 3/5 SW1-bearing mice and 5/10 B16-bearing mice vs 0/5 and 1/10, respectively, for the 3 mAb combination (p<0.05). We never observed tumor recurrence in mice that had been tumor free for >150 days after cessation of treatment and therefore consider these mice cured.

FIGURE 2.

Eradication of large SW1 and B16 tumors by i.t. injection of the 4 and 3 mAb combinations. When s.c. tumors had ~80 mm² surface area, they were injected 6 times with indicated mAbs as shown by arrows. (A) Survival curves for mice with SW1 melanoma. (B) Survival curves for mice with B16 melanoma. (C) Tumor growth curves for individual mice with s.c. SW1 melanoma (upper) or B16 melanoma (bottom). (D) Survival curves for mice bearing a SW1 melanoma transplanted on each flank, one of which was injected with the 4 mAb combination or with control; in another group, the 4 mAb combination was instead given i.p. (E) Tumor growth curves shown for both treated (right) and untreated (left) tumors in individual mice. n=5 for each group, * p<0.05, **p<0.01.

Intraperitoneal (i.p.) mAb injection is less therapeutically efficacious than i.t. injection

The mAb combinations were also administrated i.p. to mice with tumors that had a mean surface area of ~25 mm² and the data were compared with those in mice injected i.t. The 3 mAb combination induced fewer CRs when given i.p. rather than i.t. (Table 1, 5/15 versus 26/40 CR, p<0.01) to mice with SW1 tumors, as did the 4 mAb combination in the B16 model (Table 1, 1/5 versus 14/20 CR, p<0.05).

Transplanted tumor cells induce a Th2 type tumor microenvironment

SW1 or B16 cells were transplanted s.c. and the mice were euthanized after various times. Interestingly, transplanted SW1 cells significantly increased the number of CD19⁺ cells in tumor-draining lymph nodes (TLN) already after one day, and the increase persisted when last evaluated on day 21. In contrast, the number of CD3⁺ and CD8⁺ cells decreased (Fig. 3A, left panel, p<0.05). Similar results were found in C57BL/6 mice transplanted with B16 cells (Supplemental Fig. 1). Besides, there was a statistically significant increased number of CD11b⁺Gr-1⁺ MDSCs in spleens from mice transplanted with either SW1 or TC1 cells (Fig. 3A, right panel). Experiments were also performed in which mice were transplanted with syngeneic normal cells. These mice had a similar immunological pattern as naïve mice, indicating that the induced Th2 type environment was tumor-related.

FIGURE 3.

(A) Left panel shows cells in TLN from mice that were naïve or transplanted with 5×10⁵ cells from SW1 or syngeneic normal cells 1 day prior to euthanasia; middle panel shows relative mRNA expression for TLN from mice that were naïve or transplanted 2 days previously with 1×10⁵ B16 cells; right panel shows CD11b⁺Gr-1⁺ MDSC in spleens from mice that were naïve or transplanted with 5×10⁵ SW1 or TC1 cells 14 or 21 days prior to euthanasia (n=5/group). (B) Photographs of TLN (top) and spleen (bottom) from SW1-bearing mice 7 days after one injection of the 3 mAb combination. (C) Lymphocyte populations in TLN from SW1-bearing mice treated as indicated (n=5/group). (D) Lymphocyte populations in spleens from the same mice (n=5/group). (E) Dot plots and numbers of CD8⁺ (upper) and CD4⁺ (bottom) spleen cells expressing IFNγ and TNFα after culture with Cell Stimulation Cocktail for 16 hours (n=3/group). (F) Dot plots showing decrease of CD19⁺ cells and increase of CD3⁺, CD4⁺, CD8⁺ cells and CD11c⁺CD86⁺ mature DCs in TIL from SW1-bearing mice which had been treated once with the 3 mAb combination as compared to control mice. (G) IHC images showing CD19, CD8 and CD4 cells in SW1 tumors of these mice. * p<0.05, **p<0.01, *** p<0.001. Similar results were obtained in at least three independent experiments.

To further investigate the effect of transplanted tumor cells on the immune system, RNAs were extracted from TLN of mice transplanted with B16 cells 2 days earlier and evaluated by qRT-PCR. As shown in Figure 3A middle panel, transplanted B16 cells induced a twofold decrease in IFNγ and TNFα mRNA levels and a twofold increase in IL4 mRNA levels; the changes were statistically significant.

Combinations of immunomodulatory mAbs shift a Th2 to a Th1 immunological profile

5×10⁵ SW1 cells were transplanted s.c., mAbs were injected once i.t. to tumors with a surface area ~25 mm², and mice were euthanized 7 days later. TLNs and spleens were enlarged after injection of the 3 mAb combination as compared to control mice or mice injected with a single mAb (Fig. 3B). The 3 mAb combination dramatically increased the numbers of CD3⁺, CD8⁺, CD4⁺ cells and CD11c⁺CD86⁺ mature DC in both TLN and spleen (Fig. 3C & 3D, p<0.001). It also increased the number of cells expressing CD86 or CD137, while expression of PD-1 and CTLA4 was unaffected in both TLN and spleen (Supplemental Fig. 2).

To investigate whether there was a functional response, splenocytes from SW1-bearing mice euthanized 7 days after onset of therapy were cultured with Cell Stimulation Cocktail and intracellular cytokines measured. The 3 mAb combination increased IFNγ and TNFα double-producing CD8⁺ and CD4⁺ cells compared to control or single mAb (Fig 3E, p<0.001).

The 3 mAb combination significantly increased tumor infiltration by T cells and CD11c⁺D86⁺ mature DCs and decreased infiltration by CD19⁺ cells (Fig. 3F). Immunohistochemistry staining confirmed the decrease of infiltrating CD19⁺ cells and increase of CD8⁺ and CD4⁺ T cells (Fig. 3G).

The 3 mAb combination reversed tumor immunosuppression and activated anti-tumor immunity in responders but not in non-responders

TLNs and spleens were harvested from mice with B16 melanomas which either had completely regressed (‘regressors’) or were growing progressively (‘progressors’). The numbers of CD19⁺ cells and CD11b⁺Gr-1⁺ MDSCs were lower, and CD3⁺ cell numbers higher in regressors (Fig. 4A, left panel, p<0.001). Consistently, qRT-PCR showed a six-fold higher expression of IFNγmRNA a four-fold higher expression of TNFα mRNA and a two-fold lower expression of the Th2 cytokine IL4 in TLN from regressors (Fig. 4A, right panel), indicating that the suppressed immune response had been reversed in regressors but not in progressors. Similar differences were found in spleens with more CD3⁺ cells, fewer CD11b⁺Gr-1⁺ MDSCs and a two-fold higher expression of IFNγmRNA in regressors (Fig. 4B).

FIGURE 4.

Mice whose B16 melanoma had regressed or was growing progressively were euthanized 7 days after the third i.t. injection of the 3 mAb combination and their TLNs and spleens analyzed. (A) Left, lymphocyte components in TLN from regressors and progressors; right, q-PCR analyses of mRNA levels for cytokines in TLNs from the same experiment. (B) Left, lymphocyte components in spleens from regressors and progressors; right, q-PCR analyses of mRNA levels for cytokines in spleens from the same experiment. (C) Numbers of indicated CD8 and CD4 cells in TLN (top) and spleen (bottom) from regressors and progressors after culture with Cell Stimulation Cocktail for 16 hours. (D) Percentages and absolute numbers of CD44⁺CD62L⁻ effector memory CD8⁺ cells in TLN (top) and spleen (bottom) from regressors and progressors. (E) Left, Flow cytometry analysis of TIL from mice whose melanoma had started to regress (responders) or grew progressively (non-responders), as compared to control, 7 days after the second i.t. injection of the 3 mAb combination and controls. Right, q-PCR analyses of mRNA levels for cytokines in tumors from the same experiment. (F) Percentages and absolute numbers of CD44⁺CD62L⁻ effector memory CD8 cells in tumors from responders, non-responders and controls. n=5/group. * p<0.05, **p<0.01, ***p<0.001.

In vitro incubation of TLN and spleen cells with Cell Stimulation Cocktail showed that regressors had significantly higher numbers of CD8⁺ (p<0.001) and CD4⁺ (p<0.01) cells producing IFNγ and TNFα in both TLN and spleen (Fig. 4C). CD8 cells were analyzed for expression of CD44 and CD62L as markers of effector memory T cells. Regressors had significantly more CD44⁺CD62L⁻ cells than progressors in both TLN and spleen (Fig. 4D, p<0.01), indicating that a functional immune response was activated in regressors but not in progressors.

We next studied B16 tumors which had either started to regress (responders) or grew progressively (non-responders) in mice euthanized 7 days after the second i.t. injection of the 3 mAb combination. Analysis of TIL showed that responding tumors contained much fewer CD19⁺ cells and more CD3⁺, CD4⁺ and CD8⁺ cells compared to non-responders (Fig. 4E, left panel). Moreover, six-fold higher mRNA levels of IFNγ and TNFα, two-fold higher mRNA levels of perforin and a 15-fold increase of granzyme B were found in responding tumors when compared with non-responding ones which had similar levels as controls (Fig. 4E, right panel).

Approximately 90% of the infiltrating CD8 cells in responders were CD44⁺CD62L⁻ effector memory cells and were present in higher numbers than in non-responders or controls (Fig. 4F, p<0.001).

The 4 mAb combination induced a stronger Th1 response than the 3 mAb combination and caused rejection of a second, untreated tumor in the same mouse

We investigated the immune profiles of mice with B16 tumors 7 days after one i.t. injection of mAb(s). Anti-CD19 mAb, given alone or in combinations, depleted more than 95% of CD19⁺ cells (Supplementary Fig. 3); most of these cells were also CD20 positive (Supplementary Fig. 3). Importantly, there were much stronger local anti-tumor responses with more CD3⁺, CD4⁺, CD8⁺ and CD80⁺CD11c⁺ cells in TLN from mice whose tumors were injected with the 4 rather than 3 mAb combination (Fig 5A, left panel, p<0.05) and much fewer CD19 cells. Moreover, the 4 mAbs more effectively increased the shift to a Th1 from a Th2 response with an increased Tbx21/Gata3 ratio, higher mRNA expression of IFN-γ and lower mRNA expression of IL-4 (Fig 5A, right panel, p<0.05). Frequencies of CD44⁺CD62L⁻ effector memory cells in TLN were similar in mice whose tumors were injected with either combination but the 4 mAb combination increased the number of CD44⁺CD62L⁺ central memory CD8 T cells as compared to the 3 mAb combination (Fig. 5B, p<0.05).

FIGURE 5.

The 4 mAb combination induced a stronger anti-tumor immunity than the 3 mAb combination detected by analyzing TLN, spleen and tumors from B16-bearing mice 7 days after one i.t. injection of either mAb combination. (A) Left, TLN characterized by flow cytometry; right, TNL characterized by qRT-PCR for relative mRNA levels of indicated genes. (B) Dot plots and absolute numbers of CD44⁺CD62L⁺ CD8 cells in TLN from mice treated with 3 mAb or 4 mAb. (C) Upper bar graphs show CD8⁺ and effector memory T cells in spleen from mice treated as indicated; Lower bar graphs show the relative mRNA levels of IFNγ and IL4 in TIL from the indicated groups. (D) Dot plots showing that addition of anti-CD19 to the 3 mAb combination depletes a spleen cell population of CD19⁺CD11c⁺ DC which have a high expression of IDO. (E) Left, lymphocyte components and right, mRNA expression of TIL from mice treated as indicated. (F) Dot plots and graph showing CD4⁺Foxp3⁻/CD4⁺Foxp3⁺ ratios in tumors from mice receiving the 3 mAb or 4 mAb combination with the highest ratio in the group receiving the 4 mAb combination (n=7–10/group). * p<0.05, **p<0.01, ***p<0.001.

The 4 mAb combination also induced more CD8⁺ spleen cells and a higher frequency of CD44⁺CD62L⁻ effector memory T cells in spleens and higher mRNA expression of IFN-γ and lower mRNA expression of IL-4 in the spleen (Fig. 5C, p<0.05). To characterize the CD19 cells, spleens were harvested from SW1-bearing mice which had been injected i.t. with the 3 or 4 mAb combination. While CD19⁺ cells were not detected in mice given the 4 mAb combination, spleen cells and TLN from mice injected with the 3 mAb combination (as well as from tumor-bearing controls) contained cells expressing CD19 and CD20 as well as a small cell population which expressed CD19 together with the DC marker CD11c and containing a high level of IDO (Fig. 5D). These cells most likely were ‘tolerogenic’ DC similar to those previously detected in the mouse spleen (33, 41) and tumor draining lymph nodes and able to activate Treg via IDO (42).

Furthermore, the 4 mAb combination significantly increased the number of CD3, CD4, CD8 and NK cells among TIL as compared to the 3 mAb combination (Fig. 5E, left panel, p<0.05), and it gave a two-fold increase of IFNγ mRNA expression and a two-fold decrease of IL-13 mRNA expression in tumors as compared to the 3 mAb combination (Fig. 5E, right panel). There was an increase in the ratio of Teff (CD4⁺Foxp3⁻) to Treg (CD4⁺Foxp3⁺) cells in tumors from mice given the 3 mAb combination versus control (Fig. 5F, p<0.05) and a further increase in mice given the 4 mAb combination (Fig. 5F, p<0.05). Furthermore, addition of the anti-CD19 mAb to the 3 mAb combination increased the number of cells in tumors that expressed CD137 and CD86, while expression of PD-1, CTLA4, PD-L1 and PD-L2 was not affected (Supplemental Fig. 4).

Experiments were performed to investigate whether i.t. injection of the 4 mAb combination induces a systemic anti-tumor response and how this response compares to that in mice which, instead, were injected i.p. SW1 cells were transplanted on both sides of the back of mice. In one group of mice, the tumor in the right flank was injected with the 4 mAb combination, while in another group the 4 mAb combination was, instead, injected i.p.; there was also a group where the right tumor was injected with a control mAb. The mice were monitored for survival and both the right and left side tumors were measured. As shown in Fig. 2, panels D and E, i.t. injection of the 4 mAb combination caused rejection also of untreated tumors in the same mice, while i.p. injection was much less effective against both tumors. The experiment was repeated with similar results. We conclude that i.t. injection of the 4 mAb combination induces a strong systemic anti-tumor response which is more therapeutically efficacious than i.p. injection at both the local and systemic levels.

Modest side-effects

No evidence of toxicity was found in the 30 TC-1-bearing mice injected with the 3 mAb or 4 mAb combination. Three of 80 mice with the SW1 or B16 melanoma which were injected with the 3 mAb combination died as did 2 of 55 mice which received the 4 mAb combination. One of 20 mice injected i.p. died as compared to 4 of 115 mice injected i.t. The cause of death remains unknown, although histopathology reports showed marked diffuse extramedullary hematopoiesis in spleen and mild extramedullary hematopoiesis and random lymophocytic, plasmacytic and neutrophilic hepatitis in liver. Most of the deaths occurred10–20 days after the of mAb therapy had started. Temporary hair loss was seen in <30% of treated mice with SW1 tumors and around 5% depigmentation was observed in mice with SW1 or B16 melanoma.

DISCUSSION

We show that established mouse tumors, even when their surface area was ~80 mm², were rejected after repeated i.t. injection of a combination of 4 immunomodulatory mAbs (anti-CD137/PD-1/CTLA4/CD19) with >50% of the mice becoming long term tumor-free survivors and most likely cured. Efficacy was detected in all 3 models investigated. These findings are remarkable since CR and cure in mice with large tumors is rare (31). Previous studies in mice with ID8 ovarian carcinoma also demonstrated prolonged survival with a combination of mAbs to CD137/PD1/CTLA4 (35) and there was a high frequency of CR when mAbs to CD137/PD-1 were combined with cisplatin (36) although the frequency of CR with the 3 mAb combination was less than with the 4 mAb combination in the models now studied. Although the 3 mAb combination could induce CR, these were less frequent, particularly when the tumors were large.

There is evidence that anti-CTLA4 mAbs induce melanoma regression by depleting Treg cells in tumors via antibody dependent cellular cytotoxicity (ADCC) (43) and that CD137 stimulation enhances the anti-lymphoma activity of anti-CD20 mAbs through ADCC (44). However, we have not investigated the quantitative and qualitative changes of various cell populations as a result of mAb administration or the mechanisms by which the various lymphoid cell populations are influenced beyond showing that the anti-CD19 mAb effectively removes CD19⁺ cells from lymph nodes and spleens.

Intratumoral injection of the 4 mAb combination induced a systemic anti-tumor response that could reject a second, not-injected tumor in the same mouse and was more effective than i.p. injection. Rejection of untreated tumors in mice with two tumors after injection one of them with the 4 mAb combination was observed also in a previous study where the 3 mAb combination was not efficacious against untreated tumors (35). Systemic (i.p.) injection of the mAb combinations could also induced CR but was considerably less efficacious than i.t. injection. An advantage of i.t. over systemic injection of immunomodulatory mAbs has also been demonstrated by others, primarily as an approach to reduce systemic toxicity (20, 45, 46). To further improve its therapeutic window one may obtain a sustained high intratumoral level by entrapping the mAbs in nanoparticles (47) or by using constructs that combine immunomodulatory mAbs with tumor-targeting ones; if the targeting is efficient, the constructs may be administered systemically.

Large numbers of CD19⁺ cells accumulated in TLN within 24 hours after tumor cell transplantation and, in parallel, there was a decrease of CD8⁺ cells and of TNFα and IFNγ. Similar changes were not seen in mice transplanted with syngeneic normal cells, indicating that the changes were induced in response to the tumor, e.g. to tumor antigens, PD-L1 (18), TGFβ (48) and/or IDO (49).

Responding mice consistently displayed a Th1 profile with dramatic decrease of tumor-associated CD19⁺ cells and increase of the number of mature DC and of CD4 and CD8 cells, including long lived memory T cells and cells doubly positive for TNFα and IFNγ, an increase of FoxP3⁻CD4⁺ T effector cells and an increased transcription of Th1 genes. We detected no case where rejection was accompanied by a Th2 type tumor microenvironment or where tumors grew progressively in a Th1 type environment. Accumulation of Th1 cells in responding tumors was greatest among TIL and TLN but also seen in spleens. These changes were most pronounced in mice receiving the 4 mAb combination but also seen with the anti-CD137/PD-1/CTLA4 combination. It is noteworthy that a Th1 tumor environment with expression of IFNγ correlates with good prognosis in patients with advanced melanoma (50).

We found no therapy resistant tumors like we had originally anticipated expecting that therapy would select cells lacking antigens as T cells targets or the ability to present such (10, 51). Maybe, successful therapy eliminated such cell variants via some bystander effect, mediated, e.g., by activated NK cells, macrophages, molecules such as TNFα or IFNγ and/or damaged vasculature. Such bystander effects were seen in a related system where adding highly immunogenic K1735 melanoma cells that expressed anti-CD137scFv to an excess of cells from an antigenically unrelated, syngeneic sarcoma, Ag104, caused rejection of the Ag104 cells (34). Bystander effects have been detected in other models as well and involve cooperation between CD4⁺ and CD8⁺ cells (52).

Progressively growing mouse tumors had a similar Th2 profile among TIL and TLN including many CD19⁺ cells, whether the mice were untreated or did not respond to the mAb-based therapy, and we detected no case where tumors grew progressively in a Th1 type environment. Although >50% of tumors responded to the 4 mAb combination, others did not and consistently maintained a Th2 type microenvironment. We speculate that this may have been due to leakage of the mAbs from the tumors at the first treatment so that they reached the maximum size before the mice had to be euthanized according to IACUC regulations for our experimental protocol.

It is noteworthy that CD19⁺ cells were detected at tumor sites already within 24 hours of tumor transplantation and that the 4 mAb combination, which contains an anti-CD19 mAb, was therapeutically more efficacious than the 3 mAb combination and induced a stronger immune response according to in vitro assays. A small population of CD19⁺ cells which expressed CD11c and contained IDO was detected in tumor-bearing controls and tumor-bearers receiving the 3 mAb combination but not in mice given the 4 mAb combination. They were probably tolerogenic DC (33), and depletion of these cells may contribute to the better efficacy of 4 mAb combination with a higher population of memory/effector T cells, although most of the CD19 cells in tumor-bearing mice were probably B lymphocytes, including regulatory B cells which can play an important role as part of a Th2 response promoting tumor growth (32, 40, 53–55). However, B cell depletion has also been reported to impair CD4⁺ and CD8⁺ T cell tumor immunity to enhance tumor growth (56), and further investigation of tumor-associated CD19⁺ and CD20⁺ cells and their mechanism of action is needed.

The side-effects have been low in the 4 models we have studied to date with less than 5% lethality in addition to hair-loss and depigmentation seen in some mice. Since injection of multiple mAbs capable of modifying immunological responses contains the risk to cause serious toxicity it will be important to deliver the mAbs in the smallest doses that are effective and keep the level of systemic exposure as low as possible. Combination of the mAb treatment with approaches that increase the tumor specificity, e.g. therapeutic vaccination, may also improve efficacy over toxicity. It is encouraging that studies of the ID8 ovarian carcinoma showed that successfully treated mice were immune to tumor antigens as detected by ELIspot and CTL assays (35, 36) and that they could reject transplanted ID8 cells but not cells from an antigenically different syngeneic tumor (36).

We conclude that tumors, including large ones, can be destroyed by the host’s immune system by administering immunomodulatory mAbs that the 4 mAb combination is most successful in our hands and that CD19⁺ cells play a larger role in tumor growth and rejection than anticipated. We also conclude that i.t. injection is more efficacious than systemic (i.p) treatment to induce both a local and systemic response. Although mAb efficacy and safety profiles vary between species, we feel that i.t. injection of the 4 mAb combination should be considered for clinical ‘translation’.

Translational Relevance.

Immunomodulatory mAbs have shown efficacy both in preclinical models and cancer patients. However, complete regressions and cures are rare. We hypothesized that efficacy would improve by combining mAbs with different modes of action and injecting them intratumorally. The findings support our hypothesis by showing reversal of the immunosuppressive tumor microenvironment and long term tumor free survival in all 3 mouse models investigated including mice with melanomas whose surface area was ~80mm². A 4 mAb combination (anti-CD137/PD-1/CTLA4/CD19) was most efficacious, particularly against large tumors. Regression was always associated with a strong Th1 type response in tumor, tumor draining lymph nodes and spleen and was accompanied by severe reduction of the number of CD19 cells in tumors and draining lymph nodes. Relapse was not encountered suggesting that therapy resistant tumor cells arising via immunoediting were deleted by some bystander effect of the immune response. An analogous approach should be considered for human cancers.

Abbreviations

ADCC

antibody dependent cellular cytotoxicity

CR

complete regression

DC

dendritic cell

i.p

intraperitoneally

i.t

intratumoral

mAb

monoclonal antibodies

MDSC

myeloid derived suppressor cell

OS

overall survival

s.c

subcutaneous

TIL

tumor infiltrating lymphoid cell

TLN

tumor draining lymph node

Treg

regulatory T cell