PRECISION TOOLS

Unraveling the Microbiome’s Complex Role in Immunotherapy

by Randi Lundberg, DVM, Ph.D.

growing body of evidence has shown that the microbiome influences both the development of disease and patient response to therapy. In the field of immuno- oncology, new insights are coming to the forefront regarding the microbiome’s effect on tumor growth and targeted therapeutics, including immunotherapies such as checkpoint inhibitors. Research has progressed from demonstrating a link between the microbiome and cancer therapy in mice, to supporting those findings in human patients, to opening new possibilities to explore modulation of the microbiome as a personalized medicine strategy for improving immunotherapy efficacy.

Establishing a Link

Some of the first studies to demonstrate a

connection between the microbiome and

response to cancer treatment in mice were

published in 2013. Iida et al. looked at how

the microbiome impacted the efficacy of an

immunotherapy and a platinum-based

chemotherapy in tumor-bearing mice

lacking microbiota. Treatment efficacy was

reduced in germ-free mice and those treated

with antibiotics, suggesting that the gut

microbiome was important for activating an

anti-tumor immune response in the local

tumor microenvironment1. A different study

in the same publication found similar results,

with tumor-bearing mice that were germ-free

or administered antibiotics before treatment

with the oncology drug cyclophosphamide

having impaired anti-tumor response

Investigators began to consider whether

non-responder patients’ microbiomes were

impacting their anti-tumor response. Two

studies in mice arrived at the same overall

conclusion: The microbiome can modulate

response to checkpoint inhibitor therapy,

impacting its efficacy. These studies found

that certain microbiota appear beneficial to

treatment response and others detrimental,

while the use of antibiotics to modulate the

microbiome can impact anti-tumor response.

In studying melanoma progression in mice

with different microbiota, researchers at the

University of Chicago found variations in

spontaneous anti-tumor activity. The

presence of Bifidobacterium was associated with

anti-tumor activity, and administering it orally

resulted in the same level of tumor control

seen with PDL-1 therapy3.

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and drug resistance2. Together, these two

articles articulated the necessity of an intact

commensal gut microbiota to stimulate the

innate and adaptive immune system in a way

that the immune system is in better shape to

mount an anti-tumor response during therapy.

By 2015, checkpoint inhibitors such as PD-1

blockers and PD-L1 blockers had taken center

stage as a highly promising immunotherapy

approach, shifting the research focus to

exploring how the microbiome might impact

the efficacy of this groundbreaking therapy.

Checkpoint inhibitors, which work by

preventing cancer cells from switching off T

cells’ anti-cancer activity, have proven very

effective in some patients. Yet, for a subset of

the cancer patient population this treatment

approach has failed to work.

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Similarly, French researchers studied whether

the efficacy of Ipilimumab (Yervoy®) in

blocking CTLA-4 would be affected by the

composition of the microbiome in mice

bearing melanoma tumors. Both the germ-free

mice and those treated first with antibiotics

did not respond to the CTLA-4 checkpoint

blockade, while the treatment was more

efficacious in mice and patients carrying

bacteria species such as Bacteroides

thetaiotaomicron and B. fragilis4.

From Mice to Men

More recently, three studies in humans have

supported these findings in mice. Published in

November 2017 and January 2018 in Science,

the studies focused on variations in the

microbiomes of patients who respond

well to checkpoint inhibitor therapy vs.

those who do not. The three studies

demonstrated similar results across institutions

in two countries, multiple tumor types and a

variety of patient cohorts. Overall, responders

to immunotherapy displayed different

compositional and functional features of

their gut microbiomes than non-responders,

and fecal microbiota transplantation (FMT)

from patients to germ-free mice identified a

causal relationship between the microbiome

and response to immunotherapy.

The focus for researchers at the Gustav Roussy

Cancer Campus in Villejuif, France was to

review data on patients with non-small cell

lung, kidney or bladder cancer who received

treatment with a PD-1 checkpoint inhibitor.

Those who had taken antibiotics before or

soon after the start of treatment responded

less effectively to anti-PD-1 therapy, with

shorter progression-free survival and overall

survival. When germ-free mice received

fecal microbiota transplants from responder

patients, the PD-1 blockers were more

efficacious, and more CXCR3+CD4+ T cells

were observed in the tumor microenvironment5.

Researchers at the University of Texas MD

Anderson Cancer Center studied the oral and

intestinal microbiota of melanoma patients

under treatment with a PD-1 blocker, observing

a correlation between effective response to

anti-PD-1 therapy and two microbiome factors:

the diversity and the composition of the gut

microbiome. Patients with low levels of

Faecalibacterium and high levels of Bacteroidales

had shorter progression-free survival, meaning

the length of time during and after therapy

that a patient lives until either worsening

of the disease or death. When mice were

given fecal transplants from responder or

non-responder patients, their response to

the anti-PD-1 therapy was similar to patients’

response, and those with microbiota from

responders displayed more CD8+ T cells in

the tumor microenvironment6.

The third recently published study, conducted

at the University of Chicago, involved patients

with metastatic melanoma who were under

treatment with PD-L1 blockers. Those who

responded better to the anti-PD-L1 therapy

had a greater abundance of eight specific

bacteria. When the researchers evaluated

the efficacy of anti-PD-L1 therapy on mice

receiving FMT from responders and non-

responders, they discovered that the PD-L1

blocker was only effective in mice receiving

FMT from responders, with five of the eight

bacterial strains associated with anti-PD-L1

in patients also found in the mice7.

What’s Next?

With a relationship established between

the microbiome and anti-tumor response,

the door now opens to exploring whether

deliberate manipulation of the microbiome

could be an effective personalized

medicine strategy for cancer patients

undergoing checkpoint inhibitor therapy

or other forms of immunotherapy.

While there is a good deal of consistency in

the research findings to date in many respects,

there remains uncertainty about which

microbiome compositions correlate best with

an immunotherapy response. There is still

more work to be done in determining which

individual bacterial agents or microbiota

compositions are most beneficial in stimulating

or enabling an effective response to

immunotherapy, and whether those

correlations vary by tumor type or immuno-

therapy type. Studying such a complex

question will prove challenging, as some

of the associated bacteria have overlapping

functionalities; they also interact with each

other and with the host’s organs, creating

greater complexity. Germ-free mice will prove

a useful tool for this endeavor, as they can

serve as a blank slate for evaluating different

strains of bacteria under different conditions.

Germ-free mice may have a role in the clinic as

well, particularly in facilitating a personalized

approach to selection of an immunotherapy

strategy. Much like FMT helped to identify a

causal relationship between the microbiome

and anti-tumor response, this technique can

be used to help make decisions on the most

efficacious therapy for a given patient. Until

valid biomarkers based on the patient’s

microbiome profile are available, a germ-free

mouse implanted with fecal matter from a

cancer patient can be used to study the efficacy

of a particular drug prior to the patient

beginning a course of treatment. Such an

approach can be personalized to an even

greater degree by combining xenografting

of patient-derived tumor tissue to immune

system-humanized mice with patient micro-

biota transplantation. This holistic approach

enables a very precise evaluation of the specific

tumor type’s response to therapy in a

humanized immune system model, taking

the patient’s microbiome profile into account.

Use of this methodology can aid in selecting

the most efficient treatment from those

currently available, as well as exploring

questions such as what might be added to

the patient’s microbiome to make the

immunotherapy treatment more effective, or

how the immunotherapy might be adjusted to

better correspond to the patient’s microbiome.

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Already, several academic and industrial

partner collaborators have announced their

plans to test various bacterial candidates in

combination with different checkpoint

inhibitor therapies in trials involving patients

with advanced metastatic melanoma or other

cancer types.

Practical Considerations

With multiple studies demonstrating a

connection between the microbiome and

immunotherapy response, it is essential to

consider the baseline microbiome of the

mouse models employed when designing

immunotherapy drug efficacy studies. To

minimize study variability that could arise

from the microbiome’s effect, several factors

should be considered and controlled where

feasible.

Ideally, mice should be sourced from the

same vendor and the same barrier or other

production location to minimize microbiome

variations. If certain features of the microbi-

ome are necessary to have in place to test

the hypothesis, e.g. the presence of certain

Bacteroides species or Faecalibacterium, these

organisms can be introduced to the mice.

This approach covers the addition of

the bacteria of interest to the baseline

microbiome, or germ-free mice can be

used to start from scratch with an entire

microbiome of interest by FMT. When FMT

studies are conducted, the recipient mouse

should be germ-free ideally; an alternative

approach is to deplete the mouse’s

microbiome with antibiotic cocktails prior

to transplantation, though this may be less

reliable since off-target effects of drug

cocktails can occur.

It is equally important to understand the

animals’ housing and diet. If individually

ventilated cages are used, for example, the

microbiome of mice in a single cage can drift

and eventually diverge from the rest of the

colony, and such differences can become more

pronounced over multiple generations. The

composition of rodent chow can vary across

lots or seasonally, and microbial content can

vary by source location and processing

methods. Sticking to the same diet batch in

studies or using purified diets instead, together

with sterilization processes, can help to

mitigate these variances. Exposure to

pathogens, commensal bacteria in the

environment or from staff, or treatment

by antibiotics also can affect the animals’

microbiome, as can stress related to

transportation, housing conditions, or

disease. Treating the drinking water with

chlorine or acids to reduce bacterial growth

has also been shown to alter the microbiota.

As research on the microbiome’s impact

on cancer therapy has evolved, the complex

role of our gut microbiota on the efficacy

of immunotherapy has come into greater

focus. While much work remains to be done,

especially to understand which bacterial

agents best correlate with an anti-tumor

response, the most recent findings open

exciting opportunities to modulate the

microbiome on an individualized basis

to improve immunotherapy efficacy and

patient outcomes.

Randi Lundberg, DVM, Ph.D, is a Field Applications

Scientist, Taconic Biosciences. Dr. Lundberg is a Doctor

of Veterinary Medicine and holds a Ph.D. in

microbiome and experimental animals from the

University of Copenhagen. In 2018, Dr. Lundberg

received the prize of honor “Industrial Researcher

of the Year” for combining scientific excellence

with applicable business solutions.

References

1. Iida, N. et al. Commensal Bacteria Control Cancer Response to Therapy by Modulating the Tumor Microenvironment. Science (80-. ). 342, 967–970 (2013).2.Viaud, S. et al. The intestinal microbiota modulates the anticancer immune effects of cyclophosphamide. Science 342, 971–6 (2013).3.Sivan, A. et al. Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy. Science (80-. ). 350, 1084–1089 (2015).4.Vetizou, M. et al. Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota. Science (80-. ). 350, 1079–1084 (2015).5.Routy, B. et al. Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors. Science eaan3706 (2017). 6.Gopalakrishnan, V. et al. Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients. Science eaan4236 (2017). 7.Matson, V. et al. The commensal microbiome is associated with anti-PD-1 efficacy in metastatic melanoma patients. Science 359, 104–108 (2018).