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Cell therapy

 

In 1968, Steven Rosenberg thought he had encountered an extremely rare case.

Rosenberg was also a resident at West Roxbury VA Hospital. The patient’s right upper abdominal pain was unbearable. Rosenberg was examined and found that he had acute cholecystitis. Nothing serious, he thought so, it would be OK to perform a cholecystectomy.

But during the operation, he found that the patient’s abdomen had a long surgical scar. Rosenberg asked him if he had undergone major surgery before. “I had cancer before,” said the patient lightly. “It was still many years ago.”

Rosenberg recalled his previous medical records and found that he had undergone surgery in this hospital 12 years ago because of cancer of the digestive tract, and that the cancer had been transferred to the liver during surgery.

The medical record writes that although doctors can remove some large tumors, they are powerless for small metastases. At that time, the doctor probably thought that the patient’s survival time had not been very long. Therefore, the patient was advised to go home and enjoy the last time of life.

However, to Rosenberg’s surprise, when he opened the next page of the medical record, he found that the patient came to the hospital again three months later, and the next one was six months later. The medical record says that the patient has returned to work after one year.

Rosenberg thinks he has only two explanations for this. First, the doctor diagnosed the mistake, which means that the patient is not a cancer patient. Although this possibility exists, the probability is not high. The second possibility is that the patient’s cancer has recovered from the spontaneous remission process.

However, the probability of this happening is even lower, because until Rosenberg met the patient, only four such patients were reported in the medical literature. There are more than tens of millions of people who suffer from cancer each year, and about one in 100,000 people are able to relieve themselves spontaneously.

Rosenberg examined the patient’s original pathology report and the tissue sample, eliminating the first possibility. The only explanation can only be that the patient experienced a spontaneous remission process. But at the time, this was indeed a very rare thing. Rosenberg was still only a residency at the time. It was undoubtedly a huge shock for him to meet such a patient: It was like magic, he had said.

In order to allow this magic to be reproduced again, Rosenberg made a sounding simple operation. He suspected that there was some kind of factor that could resist cancer in the patient’s blood. At that time, there was exactly one patient in the hospital with the same cancer who had the same blood type as the patient who spontaneously relieved it. So Rosenberg said that after obtaining the consent of the superior. Part of the blood of the patient who spontaneously relieved his illness was imported into the patient’s body.

The results can be imagined that miracles did not happen. The patient who received the blood transfusion developed rapidly and soon passed away. Although not successful, Rosenberg became very fascinated with cancer and immunology. In order to conduct further research, Rosenberg suspended the surgical training at that time and went to Harvard for a year to systematically study the knowledge of immunology.

Rosenberg was born in a traditional Jewish immigrant family. His parents escaped from Poland to New York. After the war ended, most of Rosenberg’s parents’ relatives had already died in the massacre. Rosenberg was only six years old. A postcard was sent from Europe to New York and they were told again and again that a certain relative had been killed in the massacre at Buchenwald or Auschwitz.

This is probably the worst evil in the world. Perhaps because he was too young to experience the dark side of human nature, Rosenberg knew exactly what to do in the future: become a doctor and save the lives of patients rather than harm them.

Rosenberg has always been a workaholic. When he was still young, he had thought about breaking up with Alice O’Connell, who was still his girlfriend, because he felt that love would distract him from his research.

“I like the night,” Rosenberg wrote in a book, The Transformed Cell, published in 1992. He can also recall the joy of working late at night in the lab when he was young, drinking long-cooked sticky coffee that had been on the heater for hours. The morning sun is out of the laboratory, and probably there is no satisfaction in this world that can match it.

Steven Rosenberg

 

Patient No. 67

 

When Rosenberg first arrived at NCI, people knew very little about the anti-tumor immune response. In the late 19th and early 20th centuries, Dr. William Coley of New York used Coley toxin to activate the patient’s immune system as a treatment for cancer (see: The birth of cancer immunotherapy). However, because the efficacy is difficult to repeat, and the preparation process is complicated, Coley toxin gradually fades out of sight after chemotherapy and radiation therapy.

In fact, in the seventies that Rosenberg had just joined the work, there are still many people who do not believe that the immune response in the body can fight tumors. However, Rosenberg has always believed in the potential of cell immunotherapy. Rosenberg also did a lot of research that seems to be stupid now, such as importing pig lymphocytes into humans.

Because at that time, studies have shown that the injection of certain tumor antigens into rabbits and pigs can trigger an immune response in animals. Rosenberg believes that since there is an immune response against tumor cell antigens in animals, there may be substances in the animal’s blood that can fight tumors. But like the previous time, this time the attempt was still unsuccessful.

In 1976, Robert Gallo’s laboratory found that IL-2 stimulated the proliferation of T cells. Rosenberg hopes to apply this finding to the treatment of cancer. So he began to try to add IL-2 to the culture medium of mouse T cells. After five days, the number of T cells increased to 30 times.

IL-2 is an excellent research tool. Rosenberg also just needed this tool at the time, because his next immunotherapy program was to extract T cells from the patient’s body and then return it to the patient after amplification in vitro.

Rosenberg believes that in all T cells extracted from peripheral blood, there are always several cells that can recognize tumor cells. If such cells can be expanded, it will be possible to effectively attack the tumor cells.

 

In fact, Rosenberg and colleagues confirmed in previous experiments that after co-culturing tumor tissue with immune cells and then exposing these immune cells to other tumor tissues, these cells can kill tumor cells in vitro. Moreover, they also found that the injection of IL-2 into mice can reduce the tumor in mice.

However, the two strategies of transfusion using IL-2 cultured T cells and direct use of IL-2 failed to achieve success. What’s worse, Rosenberg found IL-2 to be very toxic and able to send every patient treated in the ICU.

 

Despite this, Rosenberg still did not give up, he began to try to use a combination of these two treatment options, which is to return to the patient in vitro cultured T cells, while using IL-2.

 

From 1980 to 1984, Rosenberg tried too many treatments, but his plans for the previous 66 patients failed. But in 1984 he met 67th patient Linda Taylor, a naval officer with melanoma.

Taylor was Rosenberg’s first patient to receive this combination therapy, and she was successfully cured. Taylor is still alive. Successful treatment by Taylor and others led Rosenberg and IL-2 therapy to the headlines of major news, and some of his NCI colleagues also began to call him Stevie Wonder.

Rosenberg then published the data. However, it was later discovered that the effect of the therapy is not so great, because the therapy can only be effective for a small part of melanoma and kidney cancer patients.

After this, Rosenberg’s laboratory began to try to extract infiltrating lymphocytes (such cells are called tumor-infiltrating lymphocytes) from surgically resected tumors, and tried to transfer them back to the patient’s body after amplification in vitro. Treatment with IL-2.

In addition to the efficacy study, the Rosenberg laboratory was still conducting a basic study of TIL: to improve the survival time of TIL cells in vivo, and this task also falls on Patrick Hwu’s body.

 

Speeding CAR

 

Patrick Hwu joined Rosenberg’s laboratory in 1989. His first job was to study the survival time of TIL cells. In order to label the T cells returned to the patient, Hwu introduced the gene encoding neomycin phosphotransferase into the genome of the T cell.

The gene expressed by this gene can protect T cells from the toxic effects of neomycin. The cells are then returned to the patient or the mouse. After a few weeks, T cells were extracted from the body and neomycin was added to the medium, after which the number of viable cells was measured.

 

The conclusion of Hwu’s study was surprising because he found that these reinfused T cells can only survive in the body for about three weeks. Some of the T cells in the human body can survive for several years, and these reintroduced T cells can only survive for three weeks. This may partly explain why TIL therapy is not effective.

However, Hwu and colleagues believe that they may be able to increase the survival time of T cells in vivo. This inspiration actually comes from bone marrow transplantation. Prior to bone marrow transplantation, patients are usually treated with myeloablative chemotherapy such as fludarabine/cyclophosphamide to destroy the patient’s immune system. In order to allow the input of immune cells to have enough space for survival.

Although this approach is theoretically feasible for TIL therapy, it is also dangerous for patients. Hwu’s colleagues are basically surgeons, and only Hwu is an oncologist who used chemotherapy drugs before. Although he is also very afraid of the patient’s problems, in a sense, he must try to improve the efficacy of TIL.

But to their surprise, the patient’s tolerance to fludarabine/cyclophosphamide was not poor. Moreover, they found that the use of IL-2 after debridement chemotherapy reduced the toxicity of IL-2 and improved the condition of some patients.

Although this method has achieved some success, the efficacy of this therapy for treating other types of tumors other than melanoma is still very poor. Why can’t this therapy be applied to other types of tumors? Hwu decided to use other means to solve this problem, such as introducing the TNF gene into TIL cells so that these cells secrete TNF after migrating to the tumor site.

However, this research project is progressing slowly. In fact, these TIL research projects conducted by Hwu are very difficult, because Hwu found that the cultivation of TIL cells in vitro was very difficult, and TIL was not as easy to express foreign genes as some other types of cells. It was able to improve the success rate of transfection at that time. The type of technology is also very limited.

 

The research project of Hwu did not succeed in the end, but his experience in cultivating TIL and introducing foreign genes into T cells had a great impact on Zelig Eshhar’s current study.

Left: Patrick Hwu Right: Zelig Eshhar

Zelig Eshhar was born in an Israeli town called Rehovot. Eshhar’s doctoral and postdoctoral studies have been focused on research in the field of immunology. All he thought in his brain was immunity, T cell structure, and antibody function. One day, Eshhar’s mind came up with a very strange idea: T-body.

When Eshhar studied the structure of the T cell receptor (TCR), he felt that there were many similarities between the TCR and the structure and function of the antibody. Antibodies are made from B cells (plasma cells) and TCR is present on T cells. Structurally, the TCR consists of alfa and beta chains, and the antibody consists of a heavy chain and a light chain, both of which contain a constant region and a variable region. Functionally speaking, both TCR and antibodies have the function of antigen recognition. Moreover, the genes that express antibodies and TCR belong to the same gene family.

However, there is a significant difference in the way TCR and antibodies recognize antigens, because antibodies can recognize the intrinsic morphology of antigens and have high affinity, but TCR can only recognize antigen fragments presented by MHC.

What happens if we replace the variable region of the TCR with the variable region of the antibody (TCR-AntiBODY, T-body)? Will the antigen specificity of the antibody be transferred to T cells?

 

The antigenic receptor of this chimeric structure also contains the constant region of TCR extracellular, as well as the transmembrane region and the intracellular structural region of TCR. Therefore, this receptor should also be able to induce T cell proliferation, mediate interleukin production and target cells. Cracking.

Eshhar also confirmed the feasibility of this idea. Eshhar’s laboratory transplanted the variable region of TNP (2,4,6-trinitrobenzene) antibody to the constant region of TCR and demonstrated that the chimeric antigen receptor (CAR) receptors are similar. The antibody is specific and binds TNP antigen in a non-MHC-restricted manner and mediates interleukin production and target cell lysis.

The purpose of Eshhar’s study was simply to verify whether the antigen specificity of the T cell receptor could be altered and mediate its corresponding cell killing effect. The idea of ​​using it in the field of cancer therapy is actually a later idea. Because there was more research on using monoclonal antibodies to treat cancer at that time, Eshhar’s technical theory could overcome the defect of TCR in recognizing tumor antigens, replacing the variable regions of TCR with antibody variable regions that target tumor antigens.

However, if the technology is applied to the treatment of diseases, Eshhar also needs to improve the above technologies. Because the immortalized cells or hybridoma cells he used before can not be directly applied to the treatment of the disease.

But Eshhar encountered difficulties in introducing chimeric genes into primary T cells. Hwu was not surprised at the difficulties encountered by Eshhar because it was very difficult for lymphocytes to express foreign genes. Hwu’s previous experience in introducing TNF genes gave him a lot of experience.

 

In order to stably express the chimeric antigen receptor gene in primary T cells, Eshhar decided to cooperate with Hwu’s laboratory. They used single-chain variable fragments (scFv) derived from antibodies to solve the problem of the stability of chimeric antigen expression.

Previous experiments have also demonstrated that the binding activity and specificity of scFv and natural Fab fragments are similar. And they used the gamma subunit of the Fc receptor to transduce the antigen-binding signal (in addition, Arthur Weiss’s lab demonstrated in 1991 that the CAR containing CD8 and the CD3 was sufficient to activate T cells).

Hwu et al. constructed a chimeric antigen receptor (CAR) acting on three different targets, one targeting breast cancer cells, one colon cancer, and one ovarian cancer. They then introduced these systems into TILs that target melanoma. Finally, they also demonstrated that CAR-T cells derived from the MOv18 antibody can effectively lyse the corresponding ovarian cancer cells.

 

In 1993, Hwu and Eshhar et al. organized the data into a paper. This article has also become a landmark article in the field of CAR-T.

But in the next decade, CAR-T therapy has experienced many hardships and setbacks.

In the 1990s, on the other side of the street where the Rosenberg laboratory was located, Carl June also studied CAR-T in the Naval Medical Research Institute in Bethesda.

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