A comprehensive review of immunotherapies in prostate cancer

Accepted Manuscript Title: A Comprehensive Review of Immunotherapies in Prostate Cancer Authors: Manuel Caitano Maia, Aaron R. Hansen PII: DOI: Reference:

S1040-8428(17)30023-9 http://dx.doi.org/doi:10.1016/j.critrevonc.2017.02.026 ONCH 2337

To appear in:

Critical Reviews in Oncology/Hematology

Received date: Revised date: Accepted date:

13-1-2017 28-2-2017 28-2-2017

Please cite this article as: Maia Manuel Caitano, Hansen Aaron R.A Comprehensive Review of Immunotherapies in Prostate Cancer.Critical Reviews in Oncology and Hematology http://dx.doi.org/10.1016/j.critrevonc.2017.02.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Review Article

A Comprehensive Review of Immunotherapies in Prostate Cancer Manuel Caitano Maia1, Aaron R. Hansen2,3

1 – Department of Medical Oncology, Instituto do Câncer do Estado de São Paulo (ICESP). Av. Dr Arnaldo, 251 – Cerqueira César – CEP 01246-000. São Paulo, Brazil. 2 – Department of Medical Oncology and Hematology, Princess Margaret Hospital. 610 University Ave, Toronto, ON, Canada. 3- Department of Medicine, University of Toronto. Medical Sciences Building, 1 King's College Cir#3172, Toronto, ON, Canada. Correspondence should be addressed to: Manuel Caitano Maia, MD Division of Medical of Oncology Instituto do Câncer do Estado de São Paulo (ICESP), Hospital das Clinicas da Faculdade de Medicina, Universidade de São Paulo. Av. Dr Arnaldo, 251, São Paulo, SP - Brazil Postal Code 01246-000 Phone: +55(11) 3893-2815; Email: [email protected]

Highlights:     

Although prostate cancer expresses many tumor associated antigens, its microenvironment is relative immunosuppressive. Sipuleucel-T is the only approved immunotherapy for prostate cancer and has driven major enthusiasm for testing new agents in this disease. Various other immune agents have been tested but failed to show clinical benefit in prostate cancer. Appropriate patient selection and trial design are crucial; and need to be tailored to account for the unique pharmacodynamics and clinical outcomes of immunotherapies. Here we review the data on completed trials and conclude with future directions, highlighting important aspects that need to be addressed to improve the evaluation of immunotherapies in prostate cancer.

Abstract Prostate cancer is the second most common malignant neoplasm in men worldwide  and the fifth cause of cancer‐related death. Although multiple new agents have been  approved for metastatic castration resistant prostate cancer over the last decade, it  is  still  an  incurable  disease.  New  strategies  to  improve  cancer  control  are  needed  and  agents  targeting  the  immune  system  have  shown  encouraging  results  in  many  tumor types. Despite being attractive for immunotherapies due to the expression of  various  tumor  associated  antigens,  the  microenvironment  in  prostate  cancer  is  relatively  immunosuppressive  and  may  be  responsible  for  the  failures  of  various  agents targeting the immune system in this disease. To date, Sipuleucel‐T is the only  immunotherapy  that  has  shown  significant  clinical  efficacy  in  this  setting,  although  the high cost and potential trial flaws have precluded its  widespread incorporation  into  clinical  practice.  Issues  with  patient  selection  and  trial  design  may  have  contributed  to  the  multiple  failures  of  immunotherapy  in  prostate  cancer  and  provides  an  opportunity  to  tailor  future  studies  to  evaluate  these  agents  more  accurately.  We  have  reviewed  all  the  completed  immune  therapy  trials  in  prostate  cancer  and  highlight  important  considerations  for  the  next  generation  of  clinical  trials.  

Keywords:  prostate  cancer;  metastatic;  castration‐resistant;  immunotherapy;  immune checkpoint inhibitor; vaccines; oncolytic viruses   

Introduction  Prostate cancer (pCa) is the second most common cancer in men, with an estimated  1.1 million patients diagnosed worldwide yearly.1 Furthermore it is the fifth leading  cause  of  cancer  mortality  in  men  accounting  for  6.6%  of  total  male  deaths.1  The  majority  of  new  cases  are  localized  or  locally  advanced,  with  20%  to  30%  of  these  patients relapsing after curative intent therapy.2, 3 Less than 5% of patients present  with de novo metastatic  pCa.2  Eight  clinical states of pCa have been described and  metastatic disease is divided into hormone sensitive (HSPC) and castrate refractory 

(CRPC) settings.4   For  men  with  metastatic  CRPC  (mCRPC),  the  median  overall  survival  (OS)  in  recent  phase 3 studies has ranged from 12.2 to 34.7 months.5‐10 Table 1 outlines the various  treatment  options  for  mCRPC  with  hormonal,  chemotherapy,  radiopharmaceutical  and  immunotherapy  agents.  These  therapies  have  demonstrated  significant  improvements  in  overall  survival  but  ultimately  metastatic  pCa  currently  remains  incurable.   Targeting the immune system  represents an appealing option  for the development  of  anticancer  treatment.  There  are  several  classes  of  immune  therapies  such  as  immune  checkpoint  inhibitors,  co‐stimulatory  antibodies,  vaccines,  adoptive  cell  transfer, tumor infiltrating lymphocytes, oncolytic viruses and cytokines. The cancer  vaccine sipuleucel‐T has been approved for use in mCRPC by the US Food and Drug  Administration  (FDA)  and  recent  early  phase  trials  of  programmed  cell  death  protein‐1  (PD‐1)  inhibitors  have  reported  promising  activity,  which  support  the  enthusiasm to develop immune therapies in pCa.5, 11, 12 Notwithstanding there have  been  several  notable  immunotherapy  failures  in  pCa  and  correlative  studies  have  demonstrated  that  the  prostate  tumor  microenvironment  is  predisposed  toward  immunosuppression.13‐15 Here we review immunotherapies that have been tested in  pCa,  highlighting  the  spectrum  of  new  agents  and  combinations.  We  also  examine  potential biomarkers and aspects of clinical trial design for immunotherapies.  

Cancer and the immune system 

  Tumors occur in the setting of a dysregulated immune system.16 The immune system  provides  several  protective  mechanisms  such  as  removing  viruses  that  can  induce  tumor  formation,  suppressing  tumorigenic  inflammatory  reactions  and  eliminating  tumor cells. Cancer initiation occurs following oncogenic cellular transformation and  failure  of  intrinsic  tumor  suppressor  processes.  Beyond  these  events  the  cancer  immunoediting concept describes 3 phases that regulate the immune system‐tumor  interaction.17  

The elimination phase involves innate and adaptive immunity removing tumor cells.  Those  tumor  cells  that  remain  enter  a  state  of  quiescence  and  exist  in  equilibrium  with  adaptive  immune  cells.  Over  time  under  the  ongoing  pressure  applied  by  the  immune  system,  tumor  cells  escape  the  equilibrium  to  proliferate  unchecked  without  regulation  by  immune  cells.18  Tables  2  and  3  summarize  the  described  concepts.     The anti‐tumor immune response is cyclical, starting with recognition of tumor neo‐ antigens by specialized antigen‐presenting cells (APCs). Subsequently, APCs present  antigen  to  effector  T  cells  and  when  this  occurs  in  the  setting  of  an  appropriate  secondary  signal,  it  leads  to  activation  of  T  cells  that  then  migrate  to  the  tumor  microenvironment  where  they  remove  cancer  cells  expressing  those  antigens.19  However,  an  intricate  network  of  stimulatory  and  inhibitory  signals  regulates  this  response that will ultimately produce ongoing immune cell activation or suppression  within  the  resultant  infiltrate  and  has  been  associated  with  patient  prognosis.  20‐24  The  type,  density  and  spatial  distribution  of  infiltrating  lymphocytes  are  strongly  correlated with survival,25‐27 which supports the development of immune therapies  to enhance antitumor immune responses.28 Figure 1 summarizes the immunoediting  process and the mechanisms of action of immune agents tested in pCa.       

Prostate Cancer Microenvironment  Observations  about  the  pCa  microenvironment  suggest  that  it  is  predominantly  immunosuppressive. The findings that support this include a low cytolytic activity of  NK  cells  within  prostate  tumor  bed,14  higher  secretion  of  TGF‐beta  by  prostate  tissue,  which  inhibits  NK  cell  and  lymphocyte  function,29  and  the  recruitment  and  accumulation of T regulatory cells (Tregs) and TH17 lymphocytes that down‐regulate  antitumor immunity.30, 31  Levels of TGF‐beta in prostate tissue are associated with high gleason scores, higher 

pathologic tumor stage and increased likelihood of post‐operative residual tumors in  localized pCa.32 Furthermore in the metastatic setting, TGF‐beta concentrations are  correlated with tumor burden.33  The  prostate  immune  microenvironment  is  dynamic,  changing  over  time,  clinical  states  and  with  treatment  exposure.  The  latter  is  characterized  by  a  series  of  phenotypic  alterations  leading  to  immunomodulation34  such  as  increased  tumor  infiltrating  lymphocytes  (TIL)  in  the  prostate  bed  following  androgen  deprivation  therapy  (ADT)35, 36  or  sensitization  of  tumor  cells  to  T‐cell  mediated  lysis  following  enzalutamide  and  abiraterone  exposure,34  and  higher  levels  of  PD‐1  ligand  (PD‐L1)  and PD‐L2 expression on enzalutamide resistant prostate cancer cells.37 

Immunotherapy in Prostate Cancer  Immunotherapy  is  focused  on  agents  developed  to  harness  the  host’s  immune  system  to  target  and  destroy  malignant  cells.  It  may  involve  various  immune  mechanisms,  such  as  stimulating  recognition  and  elimination  of  non‐self  antigens,  augmenting  or  propagating  the  antigen  presentation  process,  priming  T  cells,  enhancing  T  cell  mediated  lysis,  B  lymphocyte  activation  and  the  production  of  humoral responses.   There  are some  characteristics  that  make  pCa  attractive  for  immunotherapy.33  The  tumor  microenvironment  has  many  specific  tumor  associated  antigens  (TAAs)  such  as  prostate  specific  antigen  [PSA],  prostate  acid  phosphatase  [PAP]  and  prostate‐ specific  membrane  antigen  [PSMA].  In  addition,  a  proportion  of  tumors  have  slow  growth kinetics which provides sufficient time for the immune system to mount an  anti‐tumor  response.38,  39  Here  we  discuss  different  classes  of  immunotherapy  in  pCa,  involving  antigen‐specific  vaccination,  immune  checkpoint  inhibitors  and  immunomodulators.    In  general,  antigen‐specific  vaccination  can  be  divided  in  two  major  categories:  passive or active. Passive vaccination involves the transference of immune effectors  already activated (primed) to kill cancer cells developed ex vivo. Active vaccination is  based on agents capable of generating active T effector cells against TAAs that will 

lead to cell lysis as well as recruiting and or regulating other inflammatory cells.33, 40   Active  vaccination  is  the  main  focus  of  the  next  section  and  is  subdivided  in  two  broad components: cell‐based and vector‐based approaches. Details are provided in  Table 4. 



 

Cell‐based Vaccines   Cell‐based  vaccination  consists  of  autologous  or  allogenic  whole  cells  that  are  modified  in  order  to  induce  anti‐tumor  immune  responses.  Autologous  vaccines  activate  and  prime  the  host’s  immune  cells  for  re‐infusion,  while  allogenic  cell  vaccinations are developed by culturing tumor cell lines ex‐vivo with immunological  stimulators before infusion into patients.  Autologous Vaccines  Sipuleucel‐T is an autologous vaccine which is a personalized immunotherapy agent  processed following peripheral dendritic cell (DC) collection via leukapheresis which  is  then  incubated  in  GCS‐F  and  PAP  (PA2024)  protein.5,  40  After  a  36  to  44  hour  period,  the  primed  DC  are  re‐infused  into  the  patient  in  order  to  generate  a  PAP‐ specific CD4+ and CD8+ T cell response.33  Sipuleucel‐T was the first vaccine approved as a cancer treatment and was based on  three phase III clinical trials that assessed its efficacy in the mCRPC scenario, mainly  in  patients  with  asymptomatic  or  minimally  symptomatic  disease  with  no  visceral  metastasis. The IMPACT trial enrolled 512 patients and treated them with 4 cycles of  sipuleucel‐T.  This  study  demonstrated  an  improvement  of  4.1  months  in  OS  compared  with  placebo,  with  a  22%  reduction  of  the  risk  of  death.  Of  note,  no  difference was noted in time to objective disease progression, which was secondary  endpoint. Also, no statistically significant difference in PSA response was shown. The  reasons  for  not  improving  time  to  progression  (TTP)  are  not  fully  understood,  but  may  be  related  to  the  delayed  onset  of  anti‐tumor  immune  response  after  completion of immunotherapy 5 and is consistent with the findings from other phase 

3  trials  in  this  setting.41,  42  Safety  data  showed  the  treatment  was  overall  well  tolerated with no high frequency severe adverse events. 43  However, some authors have called attention to the fact that the improved OS seen  may  have  been  related  to  excess  harm  in  the  control  arm  due  to  leukapheresis,  especially  in  the  elderly  population  (≥65  years),  since  this  intervention  differed  between  groups.  Of  note,  the  placebo  group  had  most  of  their  circulating  lymphocytes  withdrawn  but  only  received  back  a  small  portion  of  them.  Furthermore, while ex vivo these lymphocytes were exposed to conditions that may  have  resulted  in  lysis  or  rendered  them  non‐functional.44  In  addition,  the  elderly  population  had  a  lower  than  expected  survival,  possibly  due  to  the  harmful  intervention.44  Immunologic assessment of patients treated in the IMPACT trial showed that those  who had higher antibody titers (above 400) against PA2024 benefited the most from  treatment,  with  a  higher  overall  survival.5  Perhaps  even  more  interesting  is  the  presence  of  secondary  antigen  spread  (release  of  non‐targeted  antigens)  after  sipuleucel‐T  treatment,  inducing  IgG  humoral  responses  to  non‐targeted  antigens  released  after  tumor  cell  lysis.  This  phenomena  may  act  as  a  surrogate  for  longer  OS.45 An increased benefit was also shown in subgroups of earlier disease states with  less  aggressive  features  like  lower  PSA  baseline  values,  gleason  scores,  lactate  dehydrogenase (LDH) and alkaline phosphatase.5, 46, 47  Despite  its  efficacy  and  good  safety  profile,  the  use  of  sipuleucel‐T  has  not  been  widely  adopted  mainly  due  to  the  high  level  of  required  resources  and  infra‐ structure and lack of cost‐effectiveness. 48, 49   Allogenic Vaccines  GVAX  (BioSante)  is  another  type  of  cell‐based  vaccine,  which  is  composed  of  both  castrate‐sensitive  and  castrate  resistant  allogenic  prostate  cancer  cell  lines  (LNCaP  and  PC3,  respectively)  and  transduced  with  a  replication  defective  retrovirus  genetically  modified  to  bear  GM‐CSF,  resulting  in  APC  recruitment  to  the  injection  site.50, 51 Although an initial phase 1/2 trial showed promising results,52 two phase 3 

trials (VITAL‐1 and VITAL‐2) failed to show improved outcomes and were closed early  due to futility. VITAL‐1 trial randomized patients with asymptomatic mCRPC (n=626)  to GVAX or docetaxel‐prednisone and was terminated early after an interim analysis  showed a likelihood of less 30% to meet its primary endpoint of OS.53 VITAL‐2 trial  compared GVAX alone with GVAX plus docetaxel/prednisone in symptomatic mCRPC  patients  (n=408)  and  also  closed  early  due  to  an  increased  death  rate  among  patients assigned to the intervention‐group.54  These  trials  had  several  flaws,  which  may  have  contributed  to  the  disappointing  outcomes.  GVAX  had  not  been  compared  with  a  placebo  control  and  thus  moving  forward  with  a  trial  comparing  it  with  chemotherapy  in  VITAL‐1  was  premature.  Furthermore,  the  recommended  phase  2  dose  for  the  combination  of  GVAX  and  docetaxel had not been determined prior to VITAL‐2. 33, 55 

Vector‐based Vaccines  These  consist  of  genetically  engineered  nucleic  acids  that  encode  specific  TAAs  transmitted by vectors such as bacterial plasmids or viruses.     DNA‐based vaccines  DNA‐based  vaccines  can  be  incorporated  by  host  cells  and  generate  an  immune  response  by  recruiting  APCs.56  Several  early  phase  trials  of  various  DNA  vaccines  alone  or  in  combination  with  cytokines  and  growth  factors  have  been  tested  in  different  prostate  cancer  settings.  In  general  these  agents  have  modulated  the  immune  system  leading  to  T  cell  and  humoral  changes.  In  addition  these  vaccines  have had an anti‐tumor effect as evidenced by slowing the PSA doubling time.   The  most  studied  DNA  vaccine  in  prostate  cancer  to  date  has  been  pTVG‐HP,  a  plasmid,  which encodes  PAP  protein.  It  has  been  tested  in  phase  I  and  II  trials  and  shown to generate PAP‐specific T cell responses in men with biochemically recurrent  prostate  cancer,  resulting  in  an  increase  in  PSA  doubling  time  and  long‐term  T  cell  responses.57,  58  A  phase  I/II  trial  in  non‐metastatic  CRPC  patients  with  pTVG‐HP  showed an increased PSA doubling time from 6.5 months to 9.3 months after 1 year 

follow‐up after treatment.58  Viral‐vector based vaccines  The  mechanism  of  action  involves  infection  of  epithelial  cells  that  when  lysed  liberate antigens that will be taken up by APCs and presented to CD4+ and CD8+ T  cells,  generating  the  immune  response.  In  order  to  increase  the  immunogenicity,  three  co‐stimulatory  molecules  have  been  incorporated  (B7.1,  ICAM‐1  and  LFA‐3,  also known as TRICOM).59,60  PROSTVAC‐VF is a PSA‐target pox‐virus‐based vaccine that has been developed using  a  semi‐heterologous  prime‐boost  strategy  where  sequential  administration  of  a  vaccinia  virus  prime  followed  by  a  recombinant  fowlpox‐PSA  virus  boost  is  performed.  Fowlpox  vectors  have  demonstrated  sustained  immune  response  after  an initial priming event with vaccinia‐based vaccines since they do not yield late viral  gene  products  and  thus  do  not  elicit  significant  amounts  of  host  antibodies.61  PROSTVAC‐VF  has  been  shown  to  increase  PSA  progression‐free  survival  in  63%  of  patients  for  more  than  6  months  as  well  as  significantly  slowing  the  PSA  doubling  time  from  5.3  months  to  7.7  months  in  a  phase  II  study  in  non‐metastatic  pCa  patients.62 In another phase II study with PROSTVAC‐VF, 125 patients with minimally  symptomatic  mCRPC  were  enrolled  and  randomized  to  receive  the  vaccine  or  placebo. Although the study was negative for its primary endpoint (PFS), OS after 3  years  of  follow  up  was  significantly  increased  by  8.5  months  (25.1  vs  16.6  months;  HR 0.56; p=0.0061).63 A subsequent phase III study of PROSTVAC‐VF has completed  accrual and final results are awaited (NCT01322490).64   

Adenovirus type 5 (Ad5) has also been used as vector in pCa. A phase I trial tested  Ad5‐PSA  in  mCRPC  and  reported  34%  of  patients  produced  PSA  antibodies,  68%  produced an anti‐PSA T cell responses and increased PSA doubling time in 48% of the  treated  patients,65  leading  to  a  phase  II  trial  that  is  currently  underway  (NCT00583024).66   

Personalized peptide Vaccination (PPV)  To overcome the challenges of inter‐patient immunological diversity, PPV identifies 

peptides  that  are  recognized  by  the  highest  frequency  of  precursor  cytotoxic  T  lymphocytes (CTL) for each individual.  These peptides bind to  MHC‐class  I antigens  and when administered induce CTL activation and subsequent anti‐tumor response.  The  safety  of  PPV  has  been  tested  in  several  phase  I  trials  with  the  most  common  toxicity  being  an  injection‐site  reaction.67  Further  testing  in  a  phase  II  trial  of  HLA‐ A2+  or  HLA‐A24+  in  metastatic  CRPC  was  undertaken  by  randomizing  patients  to  either  PPV  plus  low‐dose  estramustine  or  standard  dose  estramustine  alone.  The  trial  demonstrated  an  increase  in  PFS  (primary  endpoint)  from  2.8  months  to  8.5  months with HR of 0.28 (95% CI, 0.14–0.61; log‐rank P = 0.0012) in the PPV group.68  A  recently  published  phase  2  trial  of  PPV  plus  metronomic  low‐dose  cyclophosphamide  compared  with  PPV  alone  showed  no  significant  differences  in  either  PFS  or  OS,  although  patients  who  developed  a  positive  immune  response  showed a longer OS regardless of treatment arm.69 A phase III, randomized, placebo‐ controlled  trial  testing  PPV  in  HLA‐A24+  patients  is  underway  in  Japan  (UMIN000011308), with an aim to recruit 333 docetaxel‐refractory mCRPC patients.  

Immune checkpoint inhibition  Checkpoint  inhibitors  are  antibodies  that  target  regulatory  or  co‐inhibitory  molecules,  such  as  cytotoxic  T‐lymphocyte  associated  protein  4  (CTLA‐4),  PD‐1  and  its ligand (PD‐L1). These checkpoints typically down‐regulate the immune system by  inhibiting T‐cell activation and promoting tolerance.38 

Anti‐CTLA‐4   Ipilimumab is  a  monoclonal  antibody  that  blocks  CTLA‐4. A  phase  III  clinical trial of  799 metastatic CRPC patients who had progressed on docetaxel‐chemotherapy were  randomized to ipilimumab or placebo after bone‐directed radiotherapy (8Gy in one  fraction).  No  significant  difference  was  noted  in  the  primary  endpoint  of  OS  (11.2  months vs 10 months; HR 0.85; p=0.053), but a modest benefit was observed in PFS  with ipilimumab over placebo (4.0 vs 3.1 months, respectively; HR 0.70, p < 0.0001).  Patients in the ipilimumab arm more frequently had greater than 50% reduction in  the PSA (13.1% vs 5.3%). Additionally, a post‐hoc analysis of pre‐defined subgroups 

showed a greater benefit in patients with more favorable prognostic factors, such as  alkaline phosphatase concentration of less than 1.5 times ULN, a hemoglobin of 110  g/L or higher and no visceral metastases. In this group, median OS was 22.7months  with  ipilimumab  and  15.8  months  with  placebo  (HR  0.62;  95%CI  0.45–0.86;  p=0.0038).70  The higher clinical benefit found in favorable subgroups has been already shown in  other  immunotherapy  trials  and  reasons  may  involve  a  lower  tumor  burden  and  a  less  immunosuppressive  tumor  microenvironment.  Also,  slow  growing  tumors  may  be more likely to mount an anti‐tumor immune response.71, 72 Failure to improve OS  in the overall population could have resulted from a lack of efficacy of ipilimumab or  a sub‐optimal schedule that combined radiation with ipilimumab. Preclinical studies  have reported that higher radiation doses as well as fractionation may be preferable  when combining with immune checkpoint therapies.73‐76  A  phase  3  trial  assessing  ipilimumab  in  the  chemotherapy‐naïve  mCRPC  setting  randomized  a  total  of  400  patients  to  ipilimumab  10mg/kg  or  placebo.  Median  OS  was not significantly different between arms (28.7mo vs 29.7mo; HR 1.11; p=0.37).  However,  ipilimumab‐treated  patients  derived  a  modest  PFS  benefit  of  approximately  2  months  (5.6mo  x  3.8mo;  HR  0.67;  P  <  0.05)  and  a  higher  PSA  response  (23%  vs  8%;  p  value  not  reported).77  Interestingly,  this  trial  did  not  corroborate the subgroup analysis from the radiation and ipilimumab trial, whereby  patients with favorable prognostic factors did not perform better with the immune  therapy compared with placebo.   

Anti‐PD1/PDL‐1  The T‐cell surface molecule PD‐1 interacts with its ligand PD‐L1 (or B7‐H1) leading to  T cell inhibition. Studies have demonstrated that higher expression of PD‐L1 in tumor  infiltrating  lymphocytes  is  associated  with  poor  survival.78  Blocking  this  interaction  enhances anti‐tumor immune response. Agents targeting the PD‐1 pathway, such as  nivolumab,  pembrolizumab  and  more  recently  PD‐L1  inhibitors  such  as  atezolizumab,  have  received  regulatory  approval  in  other  tumor  types,  including 

melanoma, renal cell, lung and bladder cancer.79‐82  Currently,  there  are  many  clinical  trials  underway  exploring  the  role  of  pembrolizumab either alone or in combination with other immune therapies, such as  vaccines  or  cryosurgery,  in  HSPC  and  mCRPC  (NCT02312557,  NCT02499835,  NCT02489357,  NCT02787005),  as  well  as  a  combination  of  nivolumab  and  ipilimumab in mCRPC (NCT02601014).   Preliminary  findings  from  a  phase  1b  study  with  pembrolizumab  in  heavily  pretreated  PD‐L1  positive  advanced  prostate  cancer  patients  reported  an  ORR  of  13%, with a median duration of response (DOR) of 59 weeks and stable disease (SD)  reached  in  39%  of  enrolled  patients.11  A  phase  II  study  of  pembrolizumab  in  combination  with  enzalutamide  in  mCRPC  patients  upon  progression  on  enzalutamide alone showed  a PSA decline of ≥50% in 20% of the patients and some  of them remained progression‐free for up to 60 weeks.12

Immunomodulators  Immunomodulatory agents that target the tumor microenvironment have also been  explored  in  pCa.  Tasquinimod  (ABR‐215050;  Active  Biotech,  Lund,  Sweden),  is  a  second‐generation  quinoline‐3‐carboxamide  that  blocks  the  immunomodulatory  protein  S100A9,  which  plays  a  key  role  in  the  function  of  regulatory  myeloid  cells.  This  drug  has  been  shown  to  have  anti‐tumor  activity  in  preclinical  models  and  clinical  efficacy  in  randomized  double  blind  placebo  controlled  trial  in  pCa.83  By  modulating  regulatory  myeloid  cells,  it  decreases  immunosuppression  and  angiogenesis in the tumor microenvironment, preventing metastatic spread.84   In  a  phase  II  study  with  206  asymptomatic  or  minimally  symptomatic,  chemotherapy‐naïve  mCRPC  patients  assigned  to  tasquinimod  or  placebo,  disease  progression was significantly delayed (the primary endpoint) from 3.3 to 7.6 months  (p=0.0042) favoring the experimental therapy. Tasquinimod was reported to have an  acceptable toxicity profile.85 In a long‐term survival analysis of this trial, there was a  trend  for  longer  OS  in  the  subgroup  with  bone  only  metastasis  [34.2  versus  27.1  months (P = 0.19; HR, 0.73; 95% CI, 0.46–1.17)].86 In this regard, a phase III trial was 

initiated  which  corroborated  the  improvement  in  radiographic  PFS  found  in  the  phase II trial (7.0 vs 4.4 months; HR 0.64; p=0.001), but it failed to demonstrate an  improvement in OS (21.3 months with tasquinimod vs 24 months with placebo; HR  1.10; p=0.25).87   

Future Directions   To  date  most  immunotherapies  have  yielded  disappointing  results  in  pCa  in  comparison  with  other  tumor  types.  There  are  two  potential  strategies  to  improve  these  results.  Firstly,  the  development  of  more  effective  immune  therapies  or  rational combinations of immune treatments. Secondly, to better select patients for  these treatments using biomarkers.  

Combination Immune Therapies  The  purpose  of  combination  immune  therapies  is  to  enhance  anti‐tumor  T  cell  responses. Combinations may include dual immune therapies or immune treatments  with chemotherapy, hormonal therapy, targeted therapy, radiation or surgery. Table  5  summarizes  current  ongoing  clinical  trials  testing  combinations  involving  immunotherapies.     Androgen  ablation  has  multiple  immune  effects  and  can  modulate  cancer  cell  sensitivity  to  T  cells,  regulate  apoptotic  genes34  and  induce  thymus  regeneration,  causing  efflux  of  new  and  naive  T  cells.88  Other  data  have  shown  that  ADT  may  mitigate  self‐tolerance89  and  eliminate  tumor  cells.  Agents  targeting  the  androgen  pathway,  such  as  enzalutamide,  can  modulate  the  immune  system  to  render  pCa  cells more sensitive to immune‐mediated attack.90    In  light  of  enzalutamide’s  immunomodulatory  capacity,  it  is  an  attractive  combination with other  immune treatments. A combination of  enzalutamide  and a  vaccine targeting TWIST (an antigen involved in epithelial to mesenchymal transition  and metastasis) has already shown promising results in pre‐clinical models.90 Clinical  trials  exploring  this  effect  are  underway  with  enzalutamide  plus  the  vaccine 

PROSTVAC/TRICOM  in  mCRPC  (NCT01867333)  and  in  non‐metastatic  castration‐ sensitive pCa (NCT01875250).   Based  on  preclinical  studies  showing  that  immunotherapy  may  have  improved  efficacy  when  given  before  androgen  ablation,91  abiraterone,  a  cyp‐17  lyase  inhibitor,  is  being  evaluated  in  conjunction  or  in  sequence  with  Sipuleucel‐T  (NCT01487863). A phase II study reported the feasibility of this combination in spite  of the requirement for steroids during abiraterone use.92  Similarly,  other  combinations  of  immunotherapies  and  androgen  pathway  targeted  agents  are  being  studied,  such  as  sipuleucel‐T  with  ADT  (NCT0141391)  and  ketoconazole  in  conjunction  with  the  177  lutetium  PSMA  labelled  with  the  monoclonal antibody J591 (177Lu‐PSMA‐J591) (NCT00859781).  Preclinical data have reported that cytotoxic treatments, such as chemotherapy and  radiation,  can  produce  regulatory  T  cell  inhibition,  effector  T  and  B  cell  activation  and TAA release leading  to  immunogenic cell death.93‐95 These  findings have led to  the  development  of  clinical  trials  evaluating  various  cytotoxic  combinations:  neoadjuvant  low‐dose  cyclophosphamide  followed  by  GVAX  and  ADT  compared  to  ADT alone in localized pCa patients before prostatectomy (NCT01696877); standard  docetaxel/prednisone  chemotherapy  in  combination  with  increasing  doses  of  the  anti‐PSMA 

mAb 

177Lu‐J591 

in 

mCRPC 

patients 

(NCT00916123); 

docetaxel/prednisone alone for up to 12 cycles or the same regimen preceded by 12  weeks  of  ProstVac/PSA‐TRICOM  in  slowly  progressing  mCRPC  patients  (NCT01145508).   Immune therapies combined with other immune agents are being tested in multiple  trials.  A  phase  I  dose‐escalation  trial  of  ipilimumab  and  a  fixed  dose  of  GVAX  in  chemotherapy‐naïve mCRPC patients reported results showing this combination was  safe and well‐tolerated. Approximately 25% of patients were reported to have a PSA  decline of ≥50%.96 Another phase I dose‐escalation trial assessed the combination of  ipilimumab  and  a  poxviral  vaccine  (with  PSA‐TRICOM)  in  30  mCRPC  patients  (24  of  them  were  chemo‐naïve).  The  combination  was  well‐tolerated,  with  no  increased 

immune‐related  toxicities  compared  to  ipilimumab  alone.  Of  note,  14  (58%)  of  the  24 patients who were chemotherapy‐naïve had a PSA decline, 6 of whom had ≥50%  reduction.97  Other  combinations  of  PD‐1  checkpoint  inhibitors  with  chemotherapy,  targeted  therapy  and  hormonal  therapy  are  in  development  in  mCRPC  (NCT02861573).  

Biomarkers   To date no approved therapy for prostate cancer is selected due to the presence or  absence of a molecular aberration. While not related currently to immune therapy,  BRCA  abnormalities  are  being  utilized  to  select  patients  with  mCRPC  for  PARP  inhibitor  treatment.98  The  development  of  predictive  biomarkers  that  identify  patients  most  likely  to  respond  to  immunotherapy  is  an  area  of  active  research.  A  summary of studied immune biomarkers is provided in table 6.   

  Snyder  and  colleagues  reported  that  neo‐epitopic  signatures  predicted  anti‐tumor  response  to  CTLA‐4  blockade  with  ipilimumab  or  tremelimumab.  According  to  the  findings,  patients  with  prolonged  benefit  from  anti‐CTLA‐4  agents  shared  common  neo‐epitopes  produced  by  somatic  mutations  in  tumors.99  Other  data  have  demonstrated  that  high  mutational  load  can  be  associated  with  increased  benefit  from checkpoint inhibitors.100‐102  Another  potential  predictive  biomarker  that  has  been  explored  in  various  solid  tumors  includes  tumor  cell  or  immune  cell  PD‐L1  expression.  Several  studies  have  reported  that  patients  with  tumor  samples  that  express  PD‐L1  have  an  increased  benefit  from  anti‐PD‐1  or  anti‐PD‐L1  therapies.82,  103‐105  In  pCa  the  role  of  PD‐L1  expression  has  not  been  defined  in  relation  to  treatment  selection.  Furthermore  issues  surrounding  how  PD‐L1  positivity  is  defined,  which  tissue  should  be  tested  (primary versus metastasis) and which assay should be used remains unresolved.106‐ 109

 

The search for immune biomarkers in pCa is an active area of research and to date  no  definitive  biomarkers  have  been  found.  Nevertheless,  several  markers  appear  promising.  Most  studies  have  focused  on  measuring  immunological  activation  as  surrogates  for  clinical  benefit,  certain  genomic  alterations  like  expression  of  HLA‐ DRB1*11  or  HLA‐A*24  alleles,110  and  humoral  antigen  spread  (an  IgG  response  to  secondary antigens) after treatment exposure.45    Although  most  biomarkers  used  to  date  are  based  on  measuring  CTL  responses  to  specific  TAAs,  it  is  important  to  note  that  using  immunological  activation  as  surrogates  for  clinical  benefit  is  currently  limited,  since  assays  are  still  unreliable  because  of  lack  of  reproductibility  between  laboratories.111, 

112

  In  addition, 

evaluating  immune  parameters  in  response  to  a  specific  TAA  may  not  capture  the  role  of  epitope  spreading  or  dynamic  changes  over  time  and  with  treatment  exposure.113 The acquisition of serial biopsies is not practical, since patients may not  have  accessible  tumors,  may  not  be  willing  to  do  repeated  biopsies  and  not  all  institutions  have  radiology  services  who  can  perform  these  investigations.  Cost  considerations  involving  all  the  aforementioned  issues  must  also  be  taken  into  account.  

Considerations for clinical trial design for immunotherapy in  prostate cancer  Duration of therapy  Currently the optimal duration of immune therapy is not known. Different trials have  either treated patients until progression, or for a set period of time for example 1 to  2  years.  Addressing  this  in  a  clinical  trial  setting  is  a  challenge  and  apart  from  Checkmate‐153  (NCT02066636)  few  studies  are  designed  to  answer  this  question.  Such  a  trial  will  take  a  long  time  to  complete  and  will  require  a  large  number  of  patients.  

Endpoints  Traditional endpoints used to evaluate chemotherapy may not assess accurately the  clinical  benefit  from  immune  therapies.  This  could  reflect  that  the  mechanism  of 

action  of  most  immune  therapies  is  not  directly  cytotoxic.  Developing  an  immune  response can take several weeks and thus clinical changes may not be observed until  that has occurred. Thus TTP and PSA endpoints may not be appropriate measures of  benefit in clinical trials.33   In  recent  clinical  trials  of  immune  checkpoint  inhibitors,  an  OS  benefit  was  seen  irrespective of PFS improvements. The phase III trial of nivolumab in metastatic renal  cell carcinoma patients and the phase II study of  atezolizumab in bladder cancer are  prime  examples.81, 82  In  the  phase  III  trial  of  sipuleucel‐T  and  the  phase  II  trial  of  ProstVac VF despite no difference in PFS or TTP, an improvement in OS favoring the  experimental  arm  was  reported.  While  OS  is  the  gold  standard  endpoint  for  any  anticancer therapy, there are limitations with this endpoint especially in the first line  metastatic  setting  where  subsequent  treatments  are  likely  to  be  administered  and  thus dilute differences between the arms of the trial. Logistical considerations such  as  duration  of  study  and  cost  must  be  accounted  for  given  that  OS  endpoints  typically  prolong  the  trial  time  and  thus  increase  expense.  Ideally  surrogate  or  intermediate  endpoints  utilizing  clinical  or  pharmacodynamics  biomarkers  of  early  response or benefit could be used if they were validated. The ICECaP initiative is an  international,  multi‐institutional  collaboration  that  plans  to  identify  intermediate  clinical endpoints in patients following radiation or prostatectomy.114 While this may  not be applicable to pCa patients on immune therapies, similar efforts are needed in  patients with metastatic CRPC to aid in clinical trial design.   The conventional statistical considerations that underpin a trial may also need to be  reviewed. A delayed separation of OS curves does not comport with a proportional  hazards model and may reduce the power to detect differences between treatment  arms.115 As a result, designing clinical trials with hazard ratios as a function of time,  clearly  separating  them  before  and  after  curves  divert  could  provide  a  more  appropriate assessment of new immunotherapies in clinical trials.  

Response Assessment  RECIST criteria does not account completely for the different responses that can be 

observed with immunotherapy. To address this an immune‐related response criteria  known as ir‐RECIST was developed in order to assess response to immunotherapy.116  These criteria account for a variety of responses that range from an initial increase in  tumor  burden  before  shrinkage,  to  a  reduction  in  target  lesions,  or  slow,  steady  decline  in  total  tumor  volume,  or  reduction  in  total  tumor  burden  after  the  appearance  of  new  lesions.  This  revised  response  criteria  is  thought  to  provide  a  better evaluation of treatment response.117 

Correlative studies  Early  phase  clinical  trials  should  aim  at  establishing  dose  and  safety  in  addition  to  providing  proof  of  principle  of  mechanism  of  action  and  an  understanding  of  the  biological  impact  of  the  treatment  and  immune  modulation.  Additionally,  immunological biomarkers that are surrogates for OS are needed, since OS analysis  requires  several  years.  Reproducible  and  reliable  assays  are  needed  to  develop  effective  immune  biomarkers  that  can  be  used  in  clinical  trials.  Barriers  to  such  biomarkers and correlative studies include cost, laboratory expertise, need for serial  sampling of tumor tissue or blood and analytical validation of assays.  

Patient Selection  Ideal candidates for immunotherapy trials in pCa are those with early disease states,  low tumor burden and slow growing tumors, who have sufficient time to mount an  anti‐tumor  immune  response.  Traditionally  experimental  therapies  are  often  first  tested  in  patients  with  advanced  treatment  refractory  disease.  Presumably  the  immune  tumor  interaction  changes  as  the  disease  state  changes  which  must  be  factored in when evaluating these therapies.   Studies conducted in renal cell carcinoma, colorectal and lung cancers have provided  evidence  of  the  impact  of  disease  stage  in  clinical  benefit  from  vaccines,  showing  better  outcomes  in  subgroups  of  less  advanced  disease.118‐120  In  pCa,  a  greater  benefit  was  seen  with  PROSTVAC‐VF  vaccine  among  patients  with  less  aggressive  disease  as  predicted  by  the  Halabi  nomogram.72  Similarly,  sipuleucel‐T  provided  a  higher  clinical  benefit  in  early  disease  states  and  lower  tumor  burden.5,  121,  122 

Greater  tumor  burden  is  associated  with  a  more  immunosuppressive  microenvironment,  with  greater  inhibitory  effect  on  the  immune  system.113,  123  Patient  selection  for  immunotherapy  trials  is  challenging,  given  patients  with  early  disease  states  have  multiple  effective  treatment  options.  Possible  strategies  to  circumvent  this  issue  include  administration  of  agents  before  a  local  treatment  is  applied (such as surgery or radiation) in a window of opportunity approach, focusing  on immunological or pathological endpoints that could act as surrogates for clinical  benefit for future clinical trials such as with the ICECaP initiative.  

Conclusion  Efficacious  immunotherapies  will  need  to  overcome  the immunosuppressive  milieu  in  the  tumor  microenvironment  in  patients  with  pCa.  Our  understanding  of  the  complex  interactions  between  the  immune  system  and  pCa  microenvironment  is  ever  increasing,  which  may  identify  new  targets  for  immunotherapy.  Many  clinical  trials  are  exploring  different  combinations  of  immune  therapies  with  cytotoxic  agents,  androgen  pathway  modulators,  radiotherapy  and  targeted  drugs,  to  investigate  synergistic  effects  between  these  agents  and  define  the  appropriate  sequence and dose. The design of clinical trials of immune treatments must also be  tailored  to  assess  more  accurately  the  true  effect  of  immunotherapies.  Addressing  these  issues  will  permit  the  successful  development  and  subsequent  approval  of  immunotherapies , broadening the arsenal of treatment approaches for pCa.   Funding: This research did not receive any specific grant from funding agencies in the  public, commercial, or not‐for‐profit sectors. 

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Figure 1 – The immunoediting process and immune targets of agents developed for prostate cancer treatment.

Escape

Cell Lysis

 

Legend Tregs

Tumor Cell

Normal Cell

An1gen‐presen1ng Cell/ Dendri1c Cell

T Regulatory Cell

MSDC

Myeloid‐derived Suppressor Cell

NK Cell

Natural Killer Cell

MP Macrophage

Table‐1 – Current FDA approved therapies for mCRPC.

Setting Comparator Arm (all mCRPC)

Agent Docetaxel6

Median OS

HR (p value)

-

Mitoxantrone

18.9m vs 16.5m

HR 0.76 (p=0.009)

Post-docetaxel

Mitoxantrone

15.1m vs 12.7m

HR 0.70 (p<0001)

Pre-docetaxel

Placebo + prednisone

34.7m vs 30.3m

HR 0.81 (p=0.0033)

Post-docetaxel

Placebo + prednisone

14.8m vs 10.9m

HR 0.65 (p<0.001)

Pre-docetaxel

Placebo + prednisone

32.4m vs 30.2m

HR 0.71 (p<0.001)

Post-docetaxel

Placebo + prednisone

18.4m vs 13.6m

HR 0.63 (p=0.001)

Sipuleucel-T5

Pre or postdocetaxel; asymptomatic or minimally symptomatic

Placebo

25.8m vs 21.7m

HR 0.78 (p=0.03)

Radium-2238

Symptomatic bone metastasis only

Placebo

14.0m vs 11.2m

HR 0.70 (P=0.002)

Cabazitaxel7

10

Abiraterone

9

Enzalutamide

Table 2: Types of Immune System 

Innate System Cell type Natural Killer Cells (NK)

Function

Dendritic Cells (DC)

Antigen presenting cells (APC)

Roles

Activate T cells and stimulate NK cells

Macrophages

Adaptive System

Description Immediate response to tissue damage. Crucial role on immune activation and regulation. Linked to adaptive system by antigen presenting cells.

B lymphocytes

Production of antigenspecific antibodies CD4+ (helper)

T lymphocytes

CD8+ (effector)

Tregs

Participate in the humoral response to cancer. T Helper cells recruit other imune cells, activate effector cells. Cell lysis generates more Participate in cell TAAs, relysis initiating the cancer-immunity cycle Secretion of TGFbeta and IL-10 and expression. Supress effector T Of inhibitory cells molecules (CTLA-4 and PD1). Activate B lymphocytes and macrophages

Abbreviations: TAA = tumor associated antigen; Tregs = regulatory T cell; TGF = transforming growth factor; IL = interleukin; CTLA‐4 = cytotoxic ; PD‐1 = programmed cell death.    

Table 3: Phases of Immune‐editing process.  Phases Elimination

Immune Infiltrate Cytokines Important features Macrophages, NK, IFN-gama, Innate and adaptive Dendritic Cells MICA/B, IL-12, systems play together TNF

State of dormancy. Editing in progress. Development of immunoevasive mutations. Mainly Tregs and BCL2, VEGF, Immunossupressive. Evasion MDSC, but also TGF-beta, Expression of impaired CD8+ T galectin-1, IDO, inhibitory molecules cells IL-10. (CTAL4 and PD-1). Abbreviations: NK = natural killer; IFN = interferon; IL = interleukin; TNF =  tumor necrosis factor; Treg = T regulatory cell; MDSC = myeloid derived suppressive  cell; BCL2 = B‐cell lymphoma 2; VEGF = vascular endothelial growth factor; TGF =  transforming growth factor; IDO = indoleamine 2,3‐dioxygenase; CTLA4 = cytotoxic T-lymphocyte associated protein 4; PD‐1 = programmed cell death 1.      Equilibrium

CD8+ and CD4+ T IL-2, IFN-gama. cells

Table 4: Types of cancer vaccines.  Description

Advantages

Allogenic Cell Vaccine

Derived from inactivated tumor cell lines

Easy preparation, targets various antigens, favorable safety profile

Autologous cell vaccine

Derived from host tumor cells

Target patients` own TAAs

Cost, time and laborconsuming, complexity, large amount of material required, must be cultured with cytokines and stimulants

DNA-based

Nucleic acids encoding genes for specific TAAs

Simplicity, stability, cost

pTVG‐HP  Low immunogenicity; (PAP)  need combination with proinflammatory molecules (IL-2, GM-CSF)

Viral-vector

Incorporation of genes within virus genome followed by Infection of epithelial cells that when lysed will release TAAs, which will be presented by APCs to naïve T cells.

Easy to manufacture high amount of genetic material, large experience, cheap.

Development of antibodies against viral vector coat proteins, leading to neutralization after 1st injection; low immunogenicity

Inflammatory responses caused by vectors recruit immune infiltrates to

Disadvantages

Example 

Vaccine Type

GVAX  Onyvax 

Sipuleucel‐T 

PROSTVAC  /PSA‐TRICOM  (pox + vaccinia  viruses)  Ad5‐PSA  (adenovirus) 

site. Broad propagation among APCs Personalized Peptide Vaccination

Utilizes HLAmatched CD8+ T cells Peptides based on host’s preexisting immunity

Harnesses the pre-existing immunity Direct towards specific tumor antigens

Cost, time and laborconsuming, Expensive, low immunological response

‐ 

Abbreviations: IL = interleukin; GM‐CSF = granulocyte macrophage colony stimulating fator.

Table 5. Ongoing Clinical Trals involving Immunotherapies in Prostate Cancer  Localized Prostate Cancer Clinical Trial Pha se

Population

NCT0187525 0

II

Localized castratesensitive

NCT0141391

II

Nonmetastatic

NCT0058302 4

II

NCT0085978 1

II

Experimental arm

Control Endpoints Accru al Goal

Enzalutamide Enzaluta Primary = 160mg PO daily mide decrease in + PROSTVAC 160mg tumor reSC q2w for up PO daily growth to 7 doses rate; Secondary = immune response/ impacto in PSA. Sipuleucel-T followed by ADT (leuprolide acetate 45mg SC) q6mo

ADT (leuprolid e acetate 45mg SC) q6mo followed after 12 weeks by Sipuleuce l-T

58

Primary = imune parameters ; Secondary = safety; PK; PSA response

68

Hormonerefractory localized or metastatic

Adenoviurs/PS Not Primary = A vaccine for 3 Controlle PSA DT doses qmonthly d response; Secondary = PSA response and immue response

66

Nonmetastatic castrateresistante

Ketoconazol Ketocona Primary = 400mg PO q8h zol Metastasis for 4 weeks 400mg free followed by PO q8h survival; 177Lu-J591 for 4 Secondary Infusion weeks = PSA followed response. by Placebo

140

NCT0169687 7

I/II

NCT0141391

II

Localized Cyclophospham Degarelix Prostate ide 200mg/m2 240mg 14 Adenocarcin IV followed by days prior oma GVAX ID x 5 + to surgery ADT (degarelix 240mg SC) 14 days later on the day of surgery

Nonmetastatic

Sipuleucel-T followed by ADT (leuprolide acetate 45mg SC) q6mo

ADT (leuprolid e acetate 45mg SC) q6mo followed after 12 weeks by Sipuleuce l-T

Primary = intraprosta tic CD8+ T cell infiltration ; safety Secondary = PSA response and timeto-PSA recurrence; pCR; immune responses; intraprosta ttic CD4+ and tregs infiltration ;

29

Primary = imune parameters ; Secondary = safety; PK; PSA response

68

Metastatic Prostate Cancer NCT0260101 4

II

mCRPC ARV7 positive

Nivolumab + Not Primary= Ipilimumab Controlled PSA q3w for 12 response; weeks Secondary followed by = OS, nivolumab PFS, ORR; q2w for 36 safety; rate weeks or until of AR-V7 disease conversion progression

15

NCT0249983 5

I/II

mCRPC

pTVG-HP Not Primary = vaccine ID Controlled safety; q2w x 6 and PSA pembrolizuma response; b IV q3w x 4 6-mo PFS from D1 rate;

32

or the same schedule + pembrolizuma b from day 85.

median rPFS; ORR; Secondary = immune response and parameters

NCT0248935 7

II

HormoneADT Not Primary = sensitive (degarelix)+ Controlled proportion oligometastat Pembrolizuma of PSA < ic b q3w up to 6 0.6ng/ml doses + at 12mo; prostate gland Secondary cryoablation = PD-1 and PD-L1 expression

12

NCT0278700 5

II

mCRPC Pembrolizuma Not Primary = postb 200mg IV Controlled ORR; chemotherap q3w upto Secondary y 24mo = DCR; PSA response; safety;

250

NCT0186733 II 3

NCT0132249 0

III

mCRPC

mCRPC asymptomati c or minimally symptomatic

Enzalutamide Enzalutami Primary = 160mg PO de 160mg TTP; daily + PO daily Secondary PROSTVAC = OS; PSA SC q2w then PFS; qmonthly x imune 6mo then response q3months Prostvac-VF +/- GM-CSF

Placebo

Primary = OS; Secondary = eventfree survival

76

1298

NCT0114550 II 8

mCRPC

Prostvac/TRIC Docetaxel OM Vaccine 75mg/m2 SC q15d x 5 IV q3w followed by Docetacel 75mg/m2 IV q3w

Primary = 10 OS

NCT0148786 II 3

mCRPC

Concurrent Sipuleucel- Primary = 69 Sipuleucetel-T T + cumulative

+ abiraterone 1000 mg PO daily

abiraterona 100mg PO daily (after 6 weeks of last infusion)

sipuleucelT CD54 upregulati on; Secondary = PK; imune response; safety.

UMIN000011 III 308

mCRPC postdocetaxel (HLA-A24+)

ITK-1 (peptide Placebo vaccine) q1w x 6 followed by q2w for up to 30 times;

Primary = 333 OS; Secondary = PSA response; imune correlates; safety

NCT0231255 II 7

mCRPC upon progression on Enzalutamide

Pembrolizuma Not b q3w for 4 Controlled doses + Enzalutamide 160mg PO daily continously

Primary = 28 PSA response; Secondary = OR; imune parameters ; OS;

NCT0286157 Ib/II mCRPC 3

Multi-arm with Not backbone of Controlled Pembrolizuma be 200mg IV q3w; Arm 1: With Olaparib 400mg PO BID; Arm 2: With Docetaxel 75mg IV q3w. Arm 3: With Enzalutamide 160mg PO daily;

Primary = 210 PSA response (≥50%); safety; Secondary = ORR; OS; DCR;

NCT0091612 I 3

Docetaxel Not 75mg/m2 q3w Controlled + 177LuDOTA-J591 2 infusions (2040 mCi/dose) prior to cycle

Primary = 30 MTD; Secondary = toxicity profile

mCRPC

3. Abbreviations: NCT = national clinical trial; ADT = androgen deprivatin therapy; mCRPC = metastatic castration‐resistant prostate cancer; MTD = maximum tolerated dose; PK = pharmacokinectics; PSA = prostate especific antigen; PFS = progression‐free survival; OS = overall survival; pCR = pathologic complete response; DCR = disease control rate; ORR = overall response rate; TTP = time‐ to‐progression;

Table 6: Biomarkers  Treatment AE37 polypeptide vaccination (PPV)

Biomarker

Description

HLA-DRB1*11 Better and/or HLA-A*24 immunological and alleles (positively); clinical responses (OS) Pre-existing IFN- Predicted gama (positively) immunological and TGF-beta responses (negatively) levels

Sipuleucel-T

Comment May be surrogates for increased benefits from this therapy. Needs validation.

Needs prospective Higher Increased validation eosinophils at week immunological activation, 6 post-treatment increased PCSS and trend for higher OS of Humoral (IgG) Higher OS and Importance CD4+ T cells on response to PSA durable responses activation of and LGALS3. humoral activity

Pox-viral Vaccine

PSA-specific T cell Higher OS; response; Favorable prognostic features

Anti-PD1 and anti-CTLA-4 agents

Indolent disease and less advanced states derive greater benefit from immunotherapies in general

Mutational Load

Mutational burden Higher ORR, PFS correlates with neo- and OS epitopes formation

PD-L1 expression

Level of expression in TIL or tumor cells may be quantified and cutoff level arbitrary defined

Issues with assay reproductibility; does not accurately predicts response or lack of response. Other still unknown coinhibitory molecules probably important.

Abbreviations: IFN = interferon; TGF = transforming growth fator; PCSS = prostate cancer especific survival; PSA = prostate especific antigen; PD-L1 = programmed cell death ligand 1; CTLA-4 = cytotoxic T-lymphocyte associated protein 4; TIL = tumor infiltrate lymphocyte; ORR = overall response rate; PFS = progression-free survival; OS = overall survival.