Patent Grant
10092645
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 16, 2015, is named B7H1-275US1_SL.txt and is 101,340 bytes in size.
Methods of treatment for cancer.
Radiation therapy (RT) remains the most important non-surgical treatment in the management of solid malignancies with around 50-60% of all cancer patients receiving this treatment. The inclusion of RT in treatment regimens reduces disease recurrence and improves overall survival in the majority of common cancers (1-3). Although effective, many patients suffer from local recurrence and metastatic disease.
In addition to the direct cytoreductive effect of RT, emerging evidence suggests that the generation of anti-tumor immune responses may play an important role in the effectiveness of this treatment (4, 5). RT can lead to expression of ecto-calreticulin on tumor cells as well as the release of several damage-associated molecular patterns (DAMPs) including High Mobility Group Box 1 (HMGB1) and ATP which can lead to recruitment and activation of antigen presenting cells (APCs) and priming of tumor antigen-specific T cell responses (6-10). Despite this immune-escape frequently occurs, with tumor recurrence remaining the leading cause of mortality in patients receiving RT (11). The identification and inhibition of key drivers of immunosuppression may augment anti-tumor immune responses with the potential to improve patient outcome.
New insights into treatment failure and more effective RT combination approaches are therefore urgently required.
The programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) axis is involved in the maintenance of peripheral tolerance and the modulation of acute inflammatory responses through inhibition of T cell function and through apoptosis of activated T cells (12, 13). In addition to binding PD-1, PD-L1 can also suppress T cell function through interaction with CD80 (14). Expression of PD-L1 is inducible and thought to respond to local inflammatory milieu, particularly type I and II interferon (IFN) (12, 15, 16). Although barely detectable in most normal tissues, expression of PD-L1 has been described in multiple malignancies (reviewed in (17)). Importantly, recent clinical studies with either PD-1 or PD-L1 targeting monoclonal antibodies (mAb) have demonstrated encouraging responses in patients with advanced disease (18-21).
A dosing strategy for combining radiation therapy and a PD-1 and/or PD-L1 antagonist is desired to create maximal benefit for patients suffering from cancer.
In accordance with the description, a method of treating cancer in a patient comprises
In another mode, the at least one PD-1 and/or PD-L1 antagonist is an at least one PD-1 and/or PD-L1 antibody or functional part thereof.
In one aspect, the radiation therapy is fractionated radiation therapy. In one mode, the fractionated radiation therapy comprises from 2 to 7 fractions. In another mode, the fractionated radiation therapy comprises 5 fractions.
In one embodiment, the radiation therapy fractions are administered in sequential days. In another embodiment, the radiation therapy fractions are administered on day 1, day 2, day 3, day 4, and day 5. In one embodiment, the radiation therapy comprises about 10 Gy in 5 fractions.
In one aspect, the at least one PD-1 and/or PD-L1 antagonist is administered on at least day 1 and/or on day 5. In one mode, the at least one PD-1 and/or PD-L1 antagonist is administered multiple times. For example, the at least one PD-1 and/or PD-L1 antagonist may be administered 3 times a week.
In one embodiment, the anti-PD-1 and/or PD-L1 antibody or functional part thereof is MEDI4736.
In another embodiment, the anti-PD-1 and/or anti-PD-L1 antibody or functional part thereof is pembrolizumab, nivolumab, BMS-936558, AMP-224, or MPDL3280A.
In one aspect, the cancer is melanoma, colorectal cancer, or breast cancer. In another aspect, more than one treatment cycle is performed. In a further aspect, from 2-8 treatment cycles are performed. In one mode, treatment cycles are weekly or every other week.
Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) and together with the description, serve to explain the principles described herein.
Table 1 provides a listing of certain sequences referenced in present embodiments. CDRs have been indicated in bold and underlining.
VKG
RFTISRDNAKNSLFLQMN
AFDY
WGQGTLVTVSS
VKG
RFTISRDNAKNSLYLQMN
DMDV
WGQGTTVTVSS
VKG
RFTISRDNAKNSLYLQMN
LAFDY
WGQGTLVTVSS
VKG
RFTISRDNSKNTLYLQMN
GYLDY
WGQGTLVTVSS
VKG
RFTISRDNAKNSLFLQMN
VKG
RFTISRDNSKNTLFLQMN
GCWDY
WGQGTLVTVSS
VKG
RFTISRDNAKNSLYLQMN
AFDY
WGQGTLVTVSS
VKG
RFTISRDNAKNSLYLQMN
LAFDY
WGQGTLVTVSS
PSLKS
RISIT RDTSKNQYYL
WLSPF
DYWGQ GTTLTVSS
SVLKS
RLIIS KDNSKSQVFL
NSPYYAI
DYW GQGTSVTVSS
PSLKS
RISIT RDTSKNQYYL
WLLPF
DYWGQ GTTLTVSS
ES
GVPDRFTG SGSGTDFTLT
PLT
FGAGTKL ELK
NENFKG
KATL TVDTSSTTAY
WVGDF
DYWGQ GTTLTLSS
NQKFKG
KAIL TVDRSSSTAY
L
TDWGQGTSL TVSS
NQKFKG
KATL TVDKSSRTAY
IYDGSLDY
WG QGTALTVSS
NQNFKD
KATL TADKSSNTAY
YDVDY
WGQGT TLTVSS
TDTVKG
RFTI SRDNAKNTLF
YGSFYEYYFD Y
WGQGTTLTV
I. Methods of Treatment
The present methods encompass treatment of cancer employing at least one dose or radiation therapy and at least one dose of at least one PD-1 and/or PD-L1 antagonist. For instance, a method of treating cancer in a patient may include administering at least one dose of radiation therapy and administering at least one PD-1 and/or PD-L1 antagonist, wherein the at least one PD-1 and/or PD-L1 antagonist is administered on the same day as a dose of radiation therapy or up to and including 4 days later.
The method of treating cancer may be administered once (as a single treatment cycle) or it may be administered more than once (i.e., with multiple treatment cycles), such as on a weekly, every other week, every three weeks, or every month cycle. If the method is administered more than once, it may be administered 2, 3, 4, 5, 6, 7, or 8 times, or more.
While not being bound by theory, in a range of established syngeneic tumor models we found that low doses of fractionated RT led to upregulation of tumor cell expression of PD-L1 in vivo. We show that fractionated RT delivered in combination with αPD-1 or αPD-L1 mAbs generated effective anti-tumor CD8+ responses, which improved local tumor control and long-term survival, and protected against tumor rechallenge by the induction of a tumor antigen-specific memory immune response. CD8+ T cell production of IFNγ was found to be responsible for the upregulation of PD-L1 on tumor cells following fractionated RT. Furthermore, scheduling of anti-PD-L1 mAb relative to the delivery of fractionated RT appeared to affect therapeutic outcome; with administration of the antagonist on the same day as RT or up to and including 4 days after the first dose of fractionated RT showing benefit over administration of the antagonist more than 7 days after the conclusion of radiation therapy. And, while not being bound by theory, upregulation of tumor cell PD-L1 expression in response to low doses of fractionated RT, as used routinely in the clinic, appears to be a mechanism of adaptive immunological resistance by tumor cells; with the PD-L1/PD-1 signalling axis therefore potentially contributing to RT treatment failure. Combination therapy with RT and blockade of the PD-1/PD-L1 signalling axis has the potential to overcome this resistance but, based on our preclinical studies, dosage timing impacts the treatment results, providing important new insights for translation to the clinic.
A. PD-1 and/or PD-L1 Antagonists for Use in Methods of Treatment
In one embodiment, the at least one PD-1 and/or PD-L1 antagonist for use the present methods is an anti-PD-1 and/or anti-PD-L1 antibody or functional part thereof.
In one aspect, the at least one PD-1 and/or PD-L1 antagonist is administered on the same day as a dose of radiation therapy or up to and including 4 days later. For example, in one aspect, if radiation therapy is provided on day 1 of a treatment cycle, the at least one PD-1 and/or PD-L1 antagonist may also be administered on day 1 of the treatment cycle (i.e., the same day as a dose of radiation therapy). In another aspect, if radiation therapy is provided on day 1 of a treatment cycle, the at least one PD-1 and/or PD-L1 antagonist is administered on day 5 (i.e., 4 days later). In a further aspect, the PD-1 and/or PD-L1 antagonist may be administered on day 1, day 2, day 3, day 4, day 5, day 6, and/or day 7 of a treatment cycle (including both single and multiple treatment schedules).
In one embodiment, the at least one PD-1 and/or PD-L1 antagonist is administered multiple times in a treatment cycle. In another embodiment, the at least one PD-1 and/or PD-L1 antagonist is administered 2, 3, 4, 5 or more times in a treatment cycle. For example, the antagonist may be administered three times a week for a one-week treatment cycle, or three times a week for a treatment cycle with two or more weeks as described above.
In one aspect, the antibody or functional part thereof is chosen from those disclosed in US Publication 2010/0028330, which is incorporated by reference for the teaching of these antibodies and functional parts thereof. In one embodiment, the antibody if MEDI4736.
In another embodiment, the anti-PD-1 and/or anti-PD-L1 antibody is pembrolizumab, nivolumab, BMS-936558, AMP-224, or MPDL3280A.
The antibodies or functional parts thereof are administered in therapeutically effective amounts. Generally, a therapeutically effective amount may vary with the subject's age, condition, and sex, as well as the severity of the medical condition of the subject. A therapeutically effective amount of an antibody or functional part thereof ranges from about 0.001 to about 30 mg/kg body weight, from about 0.01 to about 25 mg/kg body weight, from about 0.1 to about 20 mg/kg body weight, or from about 1 to about 10 mg/kg. The dosage may be adjusted, as necessary, to suit observed effects of the treatment. The appropriate dose is chosen based on clinical indications by a treating physician.
The antibodies may be given as a bolus dose, to maximize the circulating levels of antibodies for the greatest length of time after the dose. Continuous infusion may also be used after the bolus dose.
As used herein, the term antibody or functional part thereof is used in the broadest sense. It may be man-made such as monoclonal antibodies (mAbs) produced by conventional hybridoma technology, recombinant technology and/or a functional fragment thereof. It may include both intact immunoglobulin molecules for example a polyclonal antibody, a monoclonal antibody (mAb), a monospecific antibody, a bispecific antibody, a polyspecific antibody, a human antibody, a humanized antibody, an animal antibody (e.g. camelid antibody), chimeric antibodies, as well as portions, fragments, regions, peptides and derivatives thereof (provided by any known technique, such as, but not limited to, enzymatic cleavage, peptide synthesis, or recombinant techniques), such as, for example, immunoglobulin devoid of light chains, Fab, Fab′, F(ab′)2, Fv, scFv, antibody fragment, diabody, Fd, CDR regions, or any portion or peptide sequence of the antibody that is capable of binding antigen or epitope. In one embodiment, the functional part is a single chain antibody, a single chain variable fragment (scFv), a Fab fragment, or a F(ab′)2 fragment.
An antibody or functional part is said to be “capable of binding” a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. Antibody fragments or portions may lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody. Examples of antibody may be produced from intact antibodies using methods well known in the art, for example by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). Portions of antibodies may be made by any of the above methods, or may be made by expressing a portion of the recombinant molecule. For example, the CDR region(s) of a recombinant antibody may be isolated and subcloned into an appropriate expression vector.
In one embodiment, an antibody or functional part is a human antibody. The use of human antibodies for human therapy may diminish the chance of side effects due to an immunological reaction in a human individual against nonhuman sequences. In another embodiment, the antibody or functional part is humanized. In another embodiment, an antibody or functional part is a chimeric antibody. This way, sequences of interest, such as for instance a binding site of interest, can be included into an antibody or functional part.
In one embodiment, the antibody may have an IgG, IgA, IgM, or IgE isotype. In one embodiment, the antibody is an IgG.
B. Radiation Therapy for Use in Methods of Treatment
Radiation therapy, also known as high-dose ionizing irradiation, is a component of the present therapeutic approach.
In one mode, the radiation therapy is fractionated radiation therapy. In one embodiment, the fractionated radiation therapy comprises from 2 to 7 fractions. In another embodiment, the fractionated radiation therapy comprises from 3 to 6 fractions. In another embodiment, the fractionated radiation therapy comprises from 4 to 5 fractions. In one mode, the fractionated radiation therapy comprises 2, 3, 4, 5, 6, or 7 fractions. In one embodiment, the fractionated radiation therapy comprises 5 fractions.
In one mode, the radiation therapy fractions are administered in sequential days. In one mode, radiation therapy may include more than one dose on a day and/or doses on sequential days. In one mode, the radiation therapy fractions are administered on day 1, day 2, day 3, day 4, and day 5. In another mode, the radiation therapy comprises about 10 Gy in 5 fractions (i.e., 2 Gy on each of 5 days).
Other fractionation schedules may be employed including accelerated fractionation (treatment given in larger daily or weekly doses to reduce the number of weeks of treatment), hyperfractionation (smaller doses of radiation given more than once a day), or hypofractionation (larger doses given once a day or less often to reduce the number of treatments).
The radiation therapy may be x-rays, gamma rays, or charged particles. The radiation therapy may be external-beam radiation therapy or internal radiation therapy (also called brachytherapy). Systemic radiation therapy, using radioactive substances, such as radioactive iodine, may also be employed.
External-beam radiation therapy includes 3D conformational radiation therapy, intensity-modulated radiation therapy, image-guided radiation therapy, tomotherapy, stereotactic radiosurgery, proton therapy, or other charged particle beams.
C. Cancers for Treatment
The present methods may be used to treat a variety of types of cancer. In one aspect, the method may be used to treat melanoma, colorectal cancer, or breast cancer. In one embodiment, the breast cancer is triple negative breast cancer.
In one embodiment, the cancer is cancer is adrenocortical tumors, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, central nervous system cancer, cervical cancer, chest cancer, colon cancer, colorectal cancer, endometrial cancer, epidermoid carcinoma, esophageal cancer, eye cancer, glioblastoma, glioma, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor, gestational trophoblastic disease, head and neck, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer (such as hepatocellular carcinoma), lung cancer (including non-small cell, small cell, and lung carcinoid tumors), lymph node cancer, lymphoma, lymphoma of the skin, melanoma, mesothelioma, mouth cancer, multiple myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, pediatric malignancies, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland, sarcoma, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, throat cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancers.
Reference will now be made in detail to the present exemplary embodiments, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice disclosed herein. The embodiments are further explained in the following examples. These examples do not limit the scope of the claims, but merely serve to clarify certain embodiments. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit being indicated by the following claims.
A) Mice and Cell Lines
BALB/c and C57Bl/6 mice were obtained from Harlan, U.K. All animal experiments were approved by a local ethical committee and performed under a United Kingdom Home Office license. CT26 murine colon carcinoma cells (ATCC) and 4434 cells isolated from BRafV600E p16−/− mice (Richard Marias, Cancer Research UK, Manchester Institute) were maintained in DMEM, and 4T1 triple negative breast cancers (ATCC) maintained in RPMI-1640, supplemented with 10% FCS, 1% L-glutamine (Invitrogen, U.K.). All cell lines were routinely screened to confirm the absence of Mycoplasma contamination.
B) Tumor Therapy
Mice were inoculated sub-cutaneously (s.c.) with either 5×105 CT26, 1×105 4 T1 or 5×106 4434 cells. Irradiations were performed 7-10 days after inoculation (when tumors were at least 100 mm3) using a Pantak HF-320 320 kV x-ray unit (Gulmay Medical, U.K.). The machine was operated at 300 kV, 9.2 mA, with filtration fitted in the x-ray beam to give a radiation quality of 2.3 mm Cu half-value layer. Mice were positioned at a distance of 350 mm from the x-ray focus, where the dose rate was 0.80 Gy/min. Administration of αPD-1 (clone RMPI-14), αPD-L1 (clone 10F.9G2) (both Biolegend) or isotype control mAb (IgG2a and IgG2b respectively) commenced on day 1 of the fractionated RT cycle (unless otherwise stated) and was administered intra-peritoneally (i.p.) 3qw for up to 3 weeks at a dose of 10 mg/kg in a dose volume of 100 μl/10 g in PBS. For cellular and cytokine depletion experiments mice received either αCD8 mAb; clone YTS169 (a gift from M. Glennie, Southampton University), αCD4 mAb; clone GK1.5 (Biolegend), αAsialo-GM1 (Wako Chemicals) or αIFNγ; clone XMG1.2 (BioXcell). Peripheral blood was sampled during therapy to confirm cellular depletion. For tumor rechallenge experiments, long-term surviving (LTS) mice were implanted contralaterally with tumor cells a minimum of 100 days after previous tumor implantation. Additional control mice were also implanted to confirm tumor growth. Experimental groups contained at least 5 mice/group and are representative at least 2 independent experiments.
C) Measurement of Cytokine Production by CD8+ T Cells Isolated from Long-Term Surviving Mice
For in vitro stimulation 3.5×106 splenocytes from either LTS or control mice were cultured for 5 days in RPMI-1640 supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 1% L-glutamine, 50 μM 2-ME and 10 IU/ml human recombinant IL-2 in the presence of either 1×106 tumor cells irradiated with 50 Gy or 1 μmol/ml of the H2-Ld restricted peptides SPSYVYHQF (SEQ ID NO: 91) (AH1)/TPHPARIGL (SEQ ID NO: 92) (β-galactosidase) (Anaspec, U.K.). Experimental groups contained 3-5 mice and are representative of 2 independent experiments. After 5 days in culture, cells were restimulated at a 1:1 ratio with 50 Gy irradiated tumor cells for 16 hours in the presence of 3 ug/ml Brefeldin A (BD Pharmingen, U.K.) and 100 IU/ml human recombinant IL-2 (Chiron, NL). For FACS analysis, cells were washed and incubated with rat anti-CD16/32 (eBioscience, U.K.) to block non-specific binding and then stained with a FITC conjugated anti-CD8a mAb (eBioscience, U.K.). Cells were then fixed/permeabilized and stained for expression of IFNγ using an APC conjugated mAb (eBioscience, U.K.).
D) Tumor and Immune Cell Phenotyping by Flow Cytomtery
To obtain single cell suspensions, tumors were processed using a gentleMacs dissociator and a murine tumor dissociation kit (Miltenyi Biotec, U.K.). For analysis, non-specific binding was blocked as described above and expression of CD4, CD8 (BD Biosciences, UK), CD45, NKP46, PD-1 and PD-L1 examined by multi-parameter flow cytometry (all eBioscience unless otherwise stated).
E) In Vitro Co-cultures
Tumor cells were cultured in the presence of 20 ng/ml IFNγ and/or TNFα for 24 hours prior to evaluation of PD-L1 expression by flow cytometry as described above. For co-culture assay either resting or activated splenocytes (treated with PBS or phorbol 12-Myristate 13-Acetate and Ionomycin cell stimulation cocktail respectively, (eBioscience, UK)) were co-cultured at a 1:1 ratio with tumor cells and tumor cell expression of PD-L1 determined as described above. Silencing of IFNγR1 expression was achieved by lentiviral transduction of cells with ShRNA (cells were also transduced with non-targeting ShRNA as controls) (Thermo Scientific, UK). Measurement of splenocyte cytokine production (IFNγ and TNFα) was measured by intracellular flow cytometry as described above.
Radiation therapy has been shown to modulate the immunogenicity of tumor cells but is rarely able to generate durable therapeutic responses that lead to systemic anti-tumor immunity alone. We show that low doses of local fractionated dose RT delivered as about 10 Gy in 5 fractions leads to increased tumor cell expression of PD-L1 with elevated expression evident 1, 3 and 5 days after the last dose of RT when compared to time-matched, non-treated (NT) mice (
Given these observations we hypothesized that the immune response generated following RT may be limited through the PD-1/PD-L1 axis. Local RT delivered as about 10 Gy in 5 daily fractions was found to significantly improve survival in mice bearing established CT26 tumors when compared to NT controls (
Blockade of PD-L1 also improved response to RT in mice bearing established 4T1 tumors where combined therapy significantly reduced tumor burden by 38% when compared to RT alone (
We next investigated the mechanisms underlying the long-term tumor control observed following combination RT and αPD-L1 mAb therapy. Initially colony forming assays were used to confirm that αPD-1 and αPD-L1 mAb were not acting as radiation sensitizers through direct interaction with tumor cells (
In contrast to early tumor control following combination RT/αPD-L1 mAb therapy, long term survival (LTS) was not impacted by the depletion of NK cells (70% LTS mice following treatment with RT/αPD-L1 vs. 77.8% following combination therapy with NK cell depletion) (
We next investigated whether immunological memory was generated following treatment with RT and αPD-L1 mAb. We show that LTS mice originally treated with RT and αPD-L1 mAb were able to completely reject tumors following contralateral rechallenge (
Initially we confirmed that treatment of tumor cells with a range of RT doses in vitro did not have any direct impact on expression of PD-L1 (
Given the clinical correlation between IFNγ in the tumor microenvironment and PD-L1 expression (16) and the impact of TNFα on this response (22) we evaluated the impact of these cytokines on PD-L1 in our cell lines. Co-culture of tumor cells with recombinant IFNγ leads to a significant 20 fold increase in cell surface expression of PD-L1 in vitro (
To establish whether activated immune cells could lead to an adaptive change in tumor cell expression of PD-L1 through the production of IFNγ and TNFα, WT CT26 cells and those transduced with either non-targeting ShRNA (NTC ShRNA) and IFNγR1 ShRNA were co-cultured with isolated resting (PBS-treated) and activated (phorbol 12-myristate 13-acetate (PMA) and ionomycin-treated) splenocytes (
To confirm the role of IFNγ on tumor cell expression of PD-L1 in response to RT in vivo, mice received RT in combination with αIFNγ blocking antibody (or isotype control). IFNγ blockade reduced tumor cell expression of PD-L1 by 2.6 fold in NT mice to the level observed on CT26 cells cultured in vitro; suggesting adaptive upregulation of PD-L1 occurs following implantation of tumor (
We evaluated 3 distinct combination schedules where mice bearing established CT26 tumors received a fractionated RT cycle of about 10 Gy in 5 fractions with administration of αPD-L1 mAb commencing either on day 1 of the fractionated RT cycle (schedule A), day 5 of the cycle (schedule B) or 7 days after completion of RT (schedule C) (
Analysis of tumor infiltrating CD4+ and CD8+ T cells 24 hours after the last dose of RT reveal increased expression of PD-1 when compared to time-matched NT controls (P<0.05, Mann-Whitney test) (
Taken together these data demonstrate that treatment with low dose fractionated RT leads to an acute increase in PD-1 expression on T cells and that sequential therapy where blockade of the PD-1/PD-L1 signalling axis is delayed until completion of an RT cycle may be ineffective potentially due to deletion or anergy of tumor-reactive CD8+ T cells.
Low doses of fractionated RT leads to upregulation of PD-L1 expression on tumor cells secondary to CD8+ T cell production of IFNγ. In mouse models of melanoma, colorectal and breast cancer we demonstrate that the activity of RT can be enhanced through combination with αPD-L1 mAb, leading to the generation of immunological memory in long-term surviving mice which is capable of protecting against tumor recurrence. Furthermore, our data reveal that dose scheduling may affect outcomes, with concurrent but not sequential therapy effective at improving local tumor control and survival.
This is the first preclinical study to demonstrate that fractionated RT leads to increased tumor cell expression of PD-L1 through CD8+ T cell production of IFNγ. Here, tumor cell expression of PD-L1 may act as a biomarker of a local anti-tumor response, suggesting that local RT may be sufficient to prime CD8+ T cell responses. However, whilst treatment with RT alone was unable to generate durable anti-cancer immunity, activity was found to be enhanced through mAb-mediated blockade of either PD-1 or PD-L1 suggesting that signalling through this axis may limit the immune response to RT. We saw no significant differences in overall survival between RT delivered in combination with either αPD-1 or αPD-L1, or a combination of both mAb. Given this observation it seems likely that the activity of these mAbs when delivered in combination with RT is through blockade of PD-1/PD-L1 signalling, and not mediated through the PD-L1/CD80 or PD-1/PD-L2 axes, although further experiments are needed to confirm this.
Our preclinical data reveal that the depletion of NK cells reduced local tumor control at early time points (up to 11 days post completion of RT) but did not impact on overall survival. In addition, the depletion of NK cells did not impact tumor cell expression of PD-L1 following RT suggesting their limited contribution to local IFNγ production. In contrast, whilst depletion of CD4+ T cells did not impact survival following combination therapy, substantial increases in tumor cell expression of PD-L1 post RT was observed. The depletion of CD4+ T cells impacts both helper T cell and Treg populations. Although further studies are needed to delineate the relative contributions of these sub-populations of CD4+ T cells we can speculate that CD4+ helper T cells may be dispensable for the generation of effective CD8+ T cell responses post RT/PD-L1 mAb therapy in our model and that either helper T cells, or more likely Treg, may be actively suppressing IFNγ production in the local tumor milieu. Therefore, the therapeutic potential to enhance anti-tumor responses by Treg depletion may be at least in part attenuated through enhanced tumor cell expression of PD-L1.
PD-L1 expression can be modulated by a number of cytokines including type-1 and 2 IFN, TNFα and TGFβ (16, 22, 30-32). We show that tumor cell expression of PD-L1 can be enhanced following co-culture with IFNγ and that addition of TNFα can further augment this response. However, blockade of IFNγR1 or in vivo depletion of IFNγ demonstrates dependence on IFNγ mediated signalling for upregulation of PD-L1, with TNFα alone incapable of modulating tumor cell PD-L1 expression. Interestingly, in vivo depletion of IFNγ reduces PD-L1 expression on syngeneic tumor cells, matching the expression profile of tumor cells in vitro. This suggests that the local tumor microenvironment in the absence of therapeutic intervention may foster immunologically driven tumor adaptation which may support tumor development. Similarly, in human melanocytic lesions expression of PD-L1 has been shown to co-localize with areas of CD8+ T cell infiltration and is postulated to represent an adaptive mechanism of immune escape (16). However, in our preclinical mouse models monotherapy with mAbs targeting PD-1/PD-L1 only demonstrates modest activity, suggesting that targeting this axis alone may be unlikely to bring about durable anti-tumor immune responses in all settings and underscores the requirement for a combinatorial approach.
In contrast to the use of a single 12 Gy dose (30), our results demonstrate that upregulation of PD-L1 occurs at much lower biologically effective doses involving delivery of fractionated RT using about 10 Gy in 5 daily fractions. This is an important finding given that this dose per fraction is more commonly used in routine clinical practice. Several studies have assessed the impact of RT dose fractionation; comparing single ablative doses, hypofractionated and fractionated RT, on the generation of anti-tumor immune responses and on the tumor microenvironment (4, 5, 25, 26, 33, 34). However, the results of these studies are equivocal and optimal RT delivery and the impact of RT dose fractionation on tumor cell expression of PD-L1 requires further investigation.
Previously, no research has addressed the impact of sequencing on the activity of RT and αPD-L1 mAb therapy. This is especially pertinent given the predisposition to adopting a sequenced combination regimen in the clinic as a strategy to maximize tolerability. We show that only blockade of PD-L1 at the time of RT delivery can enhance the therapeutic response in mouse models, with therapy 7 days after completion of RT no better than treatment with RT alone. In addition, our data reveal that PD-L1 expression is elevated by 24 hours after completion of a fractionated RT cycle and remains elevated for at least 7 days post completion. Moreover, we found elevated PD-1 expression on tumor infiltrating CD4+ and CD8+ T cells 24 hours post RT when compared to control cohorts. Expression of PD-1 was found to be consistently higher on CD8+ vs. CD4+ T cells. Taken together these data suggest that local fractionated RT can prime anti-tumor CD8+ T cell responses but that these may be attenuated by signalling through the PD-1/PD-L1 axis and early inhibition of this axis may generate durable effective anti-tumor responses. Further, the requirement for chronic vs. acute blockade of the PD-1/PD-L1 axis when combined with RT is unclear. We observed no difference in survival when αPD-L1 mAb was administered 3qw over 1 week concurrent with RT delivery vs. an extended schedule of up to 3 weeks. These data suggest that combination approaches with RT may permit a reduction in the duration of αPD-1/PD-L1 mAb therapy required to achieve therapeutic anti-tumor immune responses. Together these data have important implications for clinical trial design.
In summary, this study demonstrates that treatment with fractionated RT leads to upregulation of tumor cell expression of PD-L1 and that blockade of the PD-1/PD-L1 axis can enhance the immune response to fractionated RT in multiple syngeneic mouse models of cancer. Our data suggest that dose scheduling of αPD-L1 mAb with RT may generate therapeutic immune responses with the capacity to reduce tumor burden and improve survival. This therapeutic combination may be a promising approach for the treatment of many solid malignancies where RT is commonly used and translation to early phase clinical trial is clearly warranted.
Patient A has colorectal cancer. In week 1, patient A receives an effective amount of radiation therapy in five fractopms. In week 1, patient A also receives a therapeutic dose of MEDI4736 on day 1, day 3, and day 5. Patient A repeats this schedule for weeks 2, 3, 4, and 5.
Patient B has breast cancer. In week 1, patient B receives an effective amount of radiation therapy. In week 1, patient B also receives a therapeutic dose of MEDI4736 on day 5. Patient repeats this schedule for weeks 3, 5, and 7.
The following items provide certain embodiments disclosed herein.
Item 1. A method of treating cancer in a patient comprising
Item 2. The method of item 1, wherein the at least one PD-1 and/or PD-L1 antagonist is an at least one PD-1 and/or PD-L1 antibody or functional part thereof.
Item 3. The method of any one of items 1-2, wherein the dose of radiation therapy is about 11 Gy or lower.
Item 4. The method of item 3, wherein the dose of radiation therapy is about 10 Gy or lower.
Item 5. The method of any one of items 1-4, wherein the radiation therapy is fractionated radiation therapy.
Item 6. The method of item 5, wherein the fractionated radiation therapy comprises from 2 to 7 fractions.
Item 7. The method of item 6, wherein the fractionated radiation therapy comprises 5 fractions.
Item 8. The method of any one of items 5-7, wherein the radiation therapy fractions are administered in sequential days.
Item 9. The method of any one of items 5-8, wherein the radiation therapy fractions are administered on day 1, day 2, day 3, day 4, and day 5.
Item 10. The method of any one of items 7-9, wherein the radiation therapy comprises about 10 Gy in 5 fractions.
Item 11. The method of any one of items 1-10, wherein the at least one PD-1 and/or PD-L1 antagonist is administered on day 1.
Item 12. The method of any one of items 1-11, wherein the at least one PD-1 and/or PD-L1 antagonist is administered on day 5.
Item 13. The method of any one of items 1-12, wherein the at least one PD-1 and/or PD-L1 antagonist is administered multiple times.
Item 14. The method of any one of items 1-13, wherein the at least one PD-1 and/or PD-L1 antagonist is administered 3 times a week.
Item 15. The method of any one of items 1-14, wherein the anti-PD-1 and/or PD-L1 antibody or functional part thereof is MEDI4736.
Item 16. The method of any one of items 1-15, wherein the anti-PD-1 and/or anti-PD-L1 antibody or functional part thereof is pembrolizumab, nivolumab, BMS-936558, AMP-224, or MPDL3280A.
Item 17. The method of any one of items 1-16, wherein the cancer is melanoma, colorectal cancer, or breast cancer.
Item 18. The method of any one of items 1-17, wherein more than one treatment cycle is performed.
Item 19. The method of item 18, wherein from 2-8 treatment cycles are performed.
Item 20. The method of any one of items 18-19, wherein the treatment cycles are weekly.
Item 21. The method of any one of items 18-19, wherein the treatment cycles are every other week.
1. D. Verellen et al., Innovations in image-guided radiotherapy. Nat Rev Cancer 7, 949-960 (2007).
2. L. J. Lee et al., Innovations in radiation therapy (RT) for breast cancer. Breast 18 Suppl 3, S103-111 (2009).
3. E. Kapiteijn et al., Preoperative radiotherapy combined with total mesorectal excision for resectable rectal cancer. N Engl J Med 345, 638-646 (2001).
4. Y. Lee et al., Therapeutic effects of ablative radiation on local tumor require CD8+ T cells: changing strategies for cancer treatment. Blood 114, 589-595 (2009).
5. A. A. Lugade et al., Local radiation therapy of B16 melanoma tumors increases the generation of tumor antigen-specific effector cells that traffic to the tumor. J Immunol 174, 7516-7523 (2005).
6. L. Apetoh et al., Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med 13, 1050-1059 (2007).
7. S. J. Gardai et al., Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte. Cell 123, 321-334 (2005).
8. F. Ghiringhelli et al., Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors. Nat Med 15, 1170-1178 (2009).
9. Y. Ma et al., Anticancer chemotherapy-induced intratumoral recruitment and differentiation of antigen-presenting cells. Immunity 38, 729-741 (2013).
10. J. Honeychurch et al., Immunogenic potential of irradiated lymphoma cells is enhanced by adjuvant immunotherapy and modulation of local macrophage populations. Leuk Lymphoma 54, 2008-2015 (2013).
11. B. Cummings et al., Five year results of a randomized trial comparing hyperfractionated to conventional radiotherapy over four weeks in locally advanced head and neck cancer. Radiother Oncol 85, 7-16 (2007).
12. H. Dong et al., Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med 8, 793-800 (2002).
13. G. J. Freeman et al., Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med 192, 1027-1034 (2000).
14. M. J. Butte et al., Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses. Immunity 27, 111-122 (2007).
15. S. Spranger et al., Up-regulation of PD-L1, IDO, and T(regs) in the melanoma tumor microenvironment is driven by CD8(+) T cells. Sci Transl Med 5, 200ra116 (2013).
16. J. M. Taube et al., Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Sci Transl Med 4, 127ra137 (2012).
17. M. Sznol et al., Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer. Clin Cancer Res 19, 1021-1034 (2013).
18. J. R. Brahmer et al., Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates. J Clin Oncol 28, 3167-3175 (2010).
19. J. R. Brahmer et al., Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med 366, 2455-2465 (2012).
20. O. Hamid et al., Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma. N Engl J Med 369, 134-144 (2013).
21. E. J. Lipson et al., Durable cancer regression off-treatment and effective reinduction therapy with an anti-PD-1 antibody. Clin Cancer Res 19, 462-468 (2013).
22. A. Kondo et al., Interferon-gamma and tumor necrosis factor-alpha induce an immunoinhibitory molecule, B7-H1, via nuclear factor-kappaB activation in blasts in myelodysplastic syndromes. Blood 116, 1124-1131 (2010).
23. S. L. Topalian et al., Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366, 2443-2454 (2012).
24. S. L. Topalian et al., Durable Tumor Remission, and Long-Term Safety in Patients With Advanced Melanoma Receiving Nivolumab. J Clin Oncol, (2014).
25. M. Z. Dewan et al., Fractionated but not single-dose radiotherapy induces an immune-mediated abscopal effect when combined with anti-CTLA-4 antibody. Clin Cancer Res 15, 5379-5388 (2009).
26. S. J. Dovedi et al., Systemic delivery of a TLR7 agonist in combination with radiation primes durable antitumor immune responses in mouse models of lymphoma. Blood 121, 251-259 (2013).
27. J. Honeychurch et al., Anti-CD40 monoclonal antibody therapy in combination with irradiation results in a CD8 T-cell-dependent immunity to B-cell lymphoma. Blood 102, 1449-1457 (2003).
28. B. C. Burnette et al., The efficacy of radiotherapy relies upon induction of type i interferon-dependent innate and adaptive immunity. Cancer Res 71, 2488-2496 (2011).
29. J. Y. Kim et al., Increase of NKG2D ligands and sensitivity to NK cell-mediated cytotoxicity of tumor cells by heat shock and ionizing radiation. Exp Mol Med 38, 474-484 (2006).
30. L. Deng et al., Irradiation and anti-PD-L1 treatment synergistically promote antitumor immunity in mice. J Clin Invest 124, 687-695 (2014).
31. J. N. Ou et al., TNF-alpha and TGF-beta counter-regulate PD-L1 expression on monocytes in systemic lupus erythematosus. Sci Rep 2, 295 (2012).
32. S. Terawaki et al., IFN-alpha directly promotes programmed cell death-1 transcription and limits the duration of T cell-mediated immunity. J Immunol 186, 2772-2779 (2011).
33. D. Schaue et al., Maximizing tumor immunity with fractionated radiation. Int J Radiat Oncol Biol Phys 83, 1306-1310 (2012).
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiments may be practiced in many ways and the claims include any equivalents thereof.
As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g., +/−5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term about may include numerical values that are rounded to the nearest significant figure.
This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/013,157, filed Jun. 17, 2014, which is incorporated by reference herein in its entirety for all purposes.
| Number | Date | Country |
|---|---|---|
| WO 2012177624 | Dec 2012 | WO |
| WO 2013190555 | Dec 2013 | WO |
| Entry |
|---|
| Zeng et al (Int J Radiat Oncol Biol Phys, 2013, 86(2): 343-349). |
| Hamid et al (NEJM, 2013, 369(2): 134-144). |
| Khan et al (OncoTargets and Therapy, 2011, 4: 137-148). |
| Chang et al (Int J Radiation Oncology Biol Phys, 2006, 66(4): 1051-1055). |
| Brahmer et al (NEJM, 2012, 366(26): 2455-2465). |
| Stewart et al (Cancer Res, 2011, 71; LB-158; Abstract). |
| Verbrugge et al (Cancer Research, 2012, 72(13): 3163-3174). |
| Strogan (Radiol Oncol, 2010, 44(1): 1-12). |
| Deng, L., et al., “Irradiation and anti-PD-L1 treatment synergistically promote antitiumor immunity in mice,” Journal of Clinical Investigation, 124(2): 687-695 (2014). |
| Dovedi, S., et al., “Acquired Resistance to Fractionated Radiotherapy Can Be Overcome by Concurrent PD-L1 Blockade,” Cancer Research, 74(19): 5458-5468 (2014). |
| Liang, H., et al., “Radiation-Induced Equilibrium is a Balance between Tumor Cell Proliferation and T Cell-Mediated Killing,” Journal of Immunology, 190: 5874-5881 (2013). |
| Zeng, J., et al., “Anti-PD-1 Blockade and Stereotactic Radiation Produce Long-Term Survival in Mice With Intracranial Gliomas,” International Journal of Radiation Oncology Biology Physics, 86(2): 343-349 (2013). |
| International Search Report dated Sep. 3, 2015, in connection with corresponding International Application No. PCT/EP2015/063552. |
| Lugade, Amit A., et al., “Local Radiation Therapy of B16 Melanoma Tumors Increases the Generation of Tumor Antigen-Specific Effector Cells That Traffic to the Tumor,” J. Immunol. 2005; 174:7516-7523. |
| Reits,m Eric A., et al., “Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy,” JEM The Rockefeller University Press, vol. 203, No. 5, May 15, 2006, 1259-1271. |
| Huang, Qian, et al., “Caspase 3-mediated stimulation of tumor cell repopulation during cancer radiotherapy,” Nature Medicine, vol. 17, No. 7, Jul. 2011, 860-867. |
| Number | Date | Country | |
|---|---|---|---|
| 20160051672 A1 | Feb 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62013157 | Jun 2014 | US |
