Can cancer cells evade hunting by macrophages through camouflage?

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Can cancer cells evade hunting by macrophages through camouflage?

Can cancer cells evade hunting by macrophages through camouflage? This article will talk about how tumor cells wear a “don’t eat me” vest and how to pretend to be “good cell” under the eyelids of macrophages.

Cancer cells wearing a “don’t eat me” vest can fool macrophages

In recent years, tumor immunotherapy has rapidly become a traffic star in the biomedical circle, and a variety of PD-1 inhibitors and CAR-T therapies have impacted traditional therapies in the trend of “after waves and before waves”.

In simple terms, immunotherapy is the formation of an immune cell force, which unlocks its anti-tumor skills after various trainings, and fights against cancer cells.

Immune cells have two branches, innate immunity and adaptive immunity.

Figure 1: Members of the adaptive immune system; image source: Engineering the Immune System (Christopher S. Bond Life Sciences Center).

The “killer T cell”, the trump card of adaptive immunity, is undoubtedly a strong and powerful general with the characteristics of “ruthless, accurate and long-lasting” main force.

With the help of PD-1 inhibitors and other equipment, “killer T cells” keenly discovered cancer cells and used “Perforin” and other “Huagu San” to decisively wipe out the enemy.

Although the “killer T cells” have been successful, some cold tumors insist on keeping them out of the moat.

The “killer T cells” who watched the fire from the other side had more than enough thoughts and lacked strength, so the generals of the immune barracks discussed to focus on the use of macrophages, a seemingly Buddha-like “pioneer” who was merciless when they shot.

The English name of macrophages is Macrophage. In Greek, “Macro” means “big” and Phage means “eat”.

As the name suggests, when patrolling the human body, if macrophages encounter alien forces such as pathogens and cancer cells, they will eat them directly by simple and rude methods.

This process is called “phagocytosis” (phagocytosis). It is similar to the popular “Pac-Man” game of the last century.

Can cancer cells evade hunting by macrophages through camouflage? Figure 2: Schematic diagram of phagocytosis; image source: Khan Academy.

Macrophages are not as powerful as T cells in killing cancer cells. However, in some tumor tissues, macrophages account for half of the total tumor weight [1].

In theory, cancer cells can also be treated with “human tactics”. “Eat” is a drop of water.

However, what is strange is that some macrophages not only neglect their duties and turn a blind eye to cancer cells, but also donate them with fine nutrients such as growth factors.

In order to investigate whether the macrophages have other problems or committed the crime of hammer dereliction, scientists have conducted decades of exploration before finding out CD47, the initiator of the macrophage mutiny.

Where is CD47 sacred? As early as 1990, Hattie Gresham’s team discovered CD47 [2]. After nearly two decades, Professor Weissman of Stanford University made a ten-year battle with CD47.

In 2009, he published an article in the journal Cell and conclusively defeated CD47, pointing out that it was the number one suspect in confusing macrophages. [3].

Specifically, macrophages have a scanning artifact called “SIRPα”. When a visitor has a CD47, a “green code” will appear to indicate safety, so the SIRPα scanner is activated and the phagocytosis suppression program is activated.

The tumor cells saw through the macrophage workflow, and cleverly put on the CD47 vest.

The simple and honest macrophages encountered cancer cells when patrolling. Under the confusion of the CD47 vest, they mistakenly regarded them as their own.

Not only did they let them go in a friendly manner, they also warmly provided various conveniences.

Can cancer cells evade hunting by macrophages through camouflage?
Figure 3: The relationship between “Don’t eat me” signal CD47 and phagocytosis; Image source: Front Oncol. 2015 Jan 29;5:7.

The above research findings are done in animals. Does CD47 have any practical effects in humans?

Professor Weissman continued to work hard and found that the survival time of patients with ovarian cancer (Ovarian Cancer), glioma (Glioma) and glioblastoma (Glioblastoma) is closely related to the amount of CD47 [4], indirectly affirming the importance of CD47.

Can cancer cells evade hunting by macrophages through camouflage? Figure 4: The correlation between CD47 mRNA levels and the prognosis of solid tumors; image source: Nat Rev Cancer, 2019. 19 (10): p. 541.

Rescue the “lost” macrophages

Now that the macrophages have been vindicated, how can scientists help the macrophages to get rid of the manipulation of cancer cells and regain their anti-cancer effects?

The easiest way is to prevent CD47-SIRPα pairing, so drugs targeting CD47/SIRPα came into being.

Drugs such as CD47/SIRPα specific antibodies can unlock the phagocytic function of macrophages on cancer cells and break the “don’t eat me” ecstasy. It is touted by experts.

CD47 has also successfully become the most important tumor immunity in the post-PD-(L)1 era. One of the star targets.

Figure 5: Regulation of SIRPα-CD47 immune checkpoint on phagocytosis; Image source: J Clin Oncol, 2019. 37(12): p. 1012-1014.

In addition to promoting phagocytosis, through the design and experiment of various “missing arms and short legs” antibodies, scientists were pleasantly surprised to find that the CD47/SIRPα antibody has the effect of “multiple birds with one stone”.

Figure 6: SIRPα-CD47 antibody; image source: Trends Immunol, 2018. 39(3): p. 173-184.

A complete CD47 antibody body is similar to Popeye, with large and robust “Fab arms” for holding SIRPα, and slightly slim “Fc legs”.

Since the CD47 antibody relies on the “Fab arm” to hook up with SIRPα to help cancer cells escape and hunt, then the “Fc leg” shouldn’t matter? What’s interesting is that antibodies that lack the “Fc leg” can’t effectively destroy tumor cells [5].

Further experiments proved that the “Fc leg” itself also has the ability to promote phagocytosis.

In addition, CD47 antibodies can also assist dendritic cells to present antigens to T cells, call in T cells to help, and work together to deal with cancer cells; it can also directly activate tumor cell apoptosis pathways and induce tumor cells to “suicide” [6] .

Figure 7: The four mechanisms of targeting CD47; image source: Crit Rev Oncol Hematol. 2020 Aug;152:103014.

It’s the mule who came out for a walk, it’s time to talk about the data.

At the beginning of 2017, the old Stanford professor Weissman was ambitious and selected five malignant childhood brain tumors for CD47 antibody testing.

These five types of brain tumors in children are big bullies in the tumor world. They are extremely lethal and lethal. Once targeted, they are almost equivalent to death sentences.

However, the old man was pleasantly surprised to find that the CD47 antibody Hu5F9-G4 can indeed inhibit the growth of these five types of brain tumors in children: glioma mice were dead in about 21 days, and after injection of high-dose Hu5F9-G4 Lived for 38 days [7].

Figure 8: CD47 antibody inhibits tumor growth in C57BL/6 hosts with immune capabilities; Image source: Sci Transl Med, 2017. 9 (381).

The animal data is so powerful that Professor Weissman’s team started the human clinical investigation in one go. Two patients with clear cell ovarian cancer and fallopian tube cancer after treatment, the tumors were 50% and 44% smaller, respectively [8].

Figure 9: CD47 antibody curative effect was observed in (A) clear cell ovarian cancer patients and (B) fallopian tube cancer patients; image source: J Clin Oncol, 2019. 37(12): p. 946-953.

Can the “camouflage” of cancer cells be cracked so easily? Seeing this, careful readers may realize a flaw: the cancer cells deliberately put on the CD47 vest in order to disguise as “self” and not be eaten by macrophages. When CD47 is used to destroy cancer cells with CD47 antibody, will the fake “self” be affected?

Many normal cells do wear CD47, a “don’t eat me” vest, but cancer cells have to insert some “eat me” signal flags (such as calreticulin).

In Hamlet’s thinking of to eat or not to eat, macrophages realized that the best solution is to use the essence of Eastern philosophy to find a balance between the signals of “eat me” and “don’t eat me”, so most of them are healthy Even without the “don’t eat me” vest, the cells can survive the loss of the “eat me” signal.

The tricky thing is that the red blood cells of humans’ important friends also have “eat me” signals, which means that drugs targeting CD47 will inevitably damage red blood cells while killing tumor cells. In fact, two clinical trials were stopped because they found a decline in red blood cells in patients.

There is always a solution to a problem. Drawing on the experience of failure, scientists started the exploration of the upgraded version of CD47 antibody 2.0 and found three main countermeasures:

The audition only recognizes the antibody of cancer cell CD47

The red blood cells are concave on both sides, the tumor cells are spherical, and the body is different. When choosing the CD47 vest, the natural taste is different.

That is to say, the CD47 on the surface of the tumor cells and the red blood cells is actually different in style. In theory, it can be found and only destroyed Antibody to tumor cell CD47.

Among the organizers of all CD47 antibody auditions, Tianjing Bio is the absolute leader. Using the fully human antibody library and phage display technology, Tianjing Biotech took a different approach and finally won the CD47 monoclonal antibody champion TJC4 through reverse screening.

Unlike other CD47 antibodies, TJC4 can recognize the unique CD47 marker of tumor cells, which cannot be fully exposed on red blood cells due to glycosylation modification, so TJC4 has weak binding force to red blood cells.

The preliminary results of TJC4 clinical phase I showed that no serious hematological adverse events occurred in patients with a single injection dose ranging from 1 mg/kg to 30 mg/kg. Tianjing Bio also won AbbVie’s $2.9 billion sponsorship with TJC4 as a seed player.

Antibody reengineering

In parallel with the audition, there is also antibody rebuilding. If you can’t pick the right one, it’s a shortcut to spend time packing the second category players.

The newly created TTI-621 by Trillium Therapeutics is an antibody fusion protein composed of the N-terminal V domain of human SIRPα and the Fc region of human IgG1. The data shows that it can minimize the binding to red blood cells, thereby minimizing anemia.

Figure 10: The combination of TTI-621 and red blood cells; image source: Trillium’s official website.

Preserve the overall situation and sacrifice senescent red blood cells

Unlike aging red blood cells, the surface of new red blood cells expresses a low “eat me” signal, so they are less sensitive to CD47 antibody-mediated phagocytosis. A

fter discovering this rule, the Forty Seven company founded by Professor Weissman developed the CD47 antibody Hu5F9-G4 and used an ingenious two-step dosing regimen.

The first step is to give the patient a starting dose of 1mg/kg to sacrifice aging red blood cells in the body.

Although it will cause temporary mild anemia, it can also stimulate the maturation of reticulocytes and compensate for differentiation into fresh young red blood cells; You can rest assured that you can use a higher dose of antibody (30 mg/kg) to achieve anti-tumor effects.

Clinical data show that Hu5F9-G4 combined with azacitidine has a good effect in the treatment of patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Among 24 MDS patients, the overall response rate reached 92%, and 50% achieved complete remission; among 22 AML patients, the overall response rate was 64%, and the complete response rate was 41%.

Combine two to win and pursue

CD47 targeted drugs are very good. After all, they are single and weak. The next step is to get like-minded partners to act together.

You must know that the predecessor PD-(L)1 summoned hundreds of allies[9] to further consolidate Fang Qiu’s status.

The clinical trials of Hu5F9-G4 combined with azacitidine have been mentioned above, let’s cite a few more examples:

1 Strong combination with other immunotherapies

CD47 antibody can present tumor antigen to T cells after blocking phagocytosis, thereby inducing T cells to chase cancer cells. Therefore, combining with T cell inhibitors (PD-(L)1) is a good idea.

A number of pre-clinical studies have shown that CD47 and PD-(L)1 inhibitors indeed cooperate happily, playing a role of one plus one greater than two.

Figure 11: The effect of CD47 and PD-1 combination therapy on tumor volume; data source: [10].

In clinical trials, CD47 and PD-(L)1 antibodies also performed well. 67% of patients received RRx-001 (CD47 antibody) and nivolumab treatment and their disease was effectively controlled (NCT02518958).

In addition, the combination therapy with rituximab (CD20 antibody) has also received preliminary clinical verification.

The clinical phase 1b study (NCT02953509) evaluated the effectiveness and safety of CD47 antibody 5F9 combined with rituximab in 22 cases of relapsed or resistant diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma: 50% of patients had objective remission, of which 36% had complete remission.

The objective response rate of patients with follicular lymphoma is 71%, which is higher than that of patients with DLBCL (40%) [11].

Figure 12: Clinical data of 5F9 combined with rituximab; Data source: N Engl J Med, 2018. 379(18): p. 1711-1721.

2 Enhancing the “eat me” signal

Since cancer cells are not eaten depending on the balance of “eat me” and “don’t eat me” signals, it is not difficult to think that enhancing the “eat me” signal can make the CD47 antibody even more powerful.

Methods to enhance the “eat me” signal include chemotherapy, cytotoxic agents, and drugs that induce apoptosis, such as the combination of Hu5F9-G4 and azacitidine mentioned above.

3 Further weaken the “don’t eat me” signal

After CD47 alone is inhibited, some cancer cells are still resistant to phagocytosis. Is the “eat me” signal too strong? CD47 inhibition is not thorough enough? Macrophages can’t reach the tumor area? Or is there something strange?

With these questions in mind, Professor Weissman speculated that it is very likely that cancer cells that the CD47 antibody cannot help have other “don’t eat me” signals.

As expected, Professor Weissman discovered two more “don’t eat me” signaling molecules: MHC1 and CD24. I have to say that cancer cells are playing routines.

Compared with normal cells, some cancer cells have more CD24[12], and the combination of CD24 and Siglec-10 on macrophages (similar to CD47 and SIRPα CP) will also release the “don’t eat me” signal: “By mixing the patient’s cancer cells and macrophages in a petri dish, and then blocking the interaction between CD24 and Siglec-10, the macrophages will devour the cancer cells like a buffet” [13].

Figure 13: CD24/Siglec-10 signaling pathway; data source: Nature, 2019. 572 (7769): p. 392-396.

Although more data is needed, it is worth scrutinizing that CD24 and CD47 seem to function in complementary ways.

Some cancers, such as blood cancer, are very sensitive to CD47 antibodies, but not so cold to CD24 inhibitors; while other cancers, such as ovarian cancer, the situation is just the opposite.

Sure enough, it was a radish and a pit.

Image source: Nat Rev Drug Discov. 2019 Sep;18(10):747; Simon Bradbrook.

The CD47-SIRPα phagocytic signaling pathway was first discovered in 1990.

It was not until 2009 that it was discovered that inhibiting CD47 could restore the ability of macrophages to engulf cancer cells [14]. In the next 10 years, more than 20 CD47 antibodies entered clinical trials, and two new “don’t eat me” signaling pathways, MHC1-LILRB1[15] and CD24-Siglec-10[12], were also discovered.

From the initial clinical data, the second-generation CD47 antibody has well controlled the “injury to the innocent”, but how effective is the overall treatment? Can it provide new treatment options for patients with PD-(L)1 non-response? Are there other fish that slipped through the “Don’t Eat Me” signal? How to scientifically find partners for CD47 antibody?

Scientists believe that these questions will be answered one by one in the near future, and antibodies such as CD47 will have more opportunities to emerge.

Reference：

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