ReadingThinkingAndWriting

Imagine a person with Stage IV Chronic Lymphocytic Leukemia, a cancer of the white blood cells. A scan of his bone marrow shows almost complete replacement of healthy cells with cancerous. The cancer is chemorefractory, meaning it survives every dose even while his normal rapidly dividing cells do not. Now, instead of the normal treatment progression and a poor prognosis, some healthy T cells are extracted from his blood. They are processed, induced to replicate, and then the large resulting colony of cells is infused back into his blood. Thirty days later, a scan reveals his bone marrow to be clear of the cancer and another 6 months later shows the same; a complete resolution to the cancer.

While I asked you to imagine, you actually don’t have to; the process, the person, and the results are real; part of a clinical trial which is still actively recruiting patients. It is not complete and the information presented in the NIH webcast, which was my original reference for this post, has not been audited let alone published₁. The same however, was not the case in all CAR trials, two courses in others, resulting instead, sadly, in fatalities₂. However, as I will present in my next post, the protocol was changed for the second group and there were special circumstances. (* See Note: I am not a physician, I am self-taught in the bio-sciences).

This new approach to cancer treatment, which includes the specific therapy that killed this man’s cancer, is known as Adoptive Cell Therapy. Specifically in this case, engineered genes coding for what is known as a Chimeric Antigen Receptor (CAR) were introduced into the T cells extracted from his blood via a retro-viral vector. I wrote about Antigen Receptors over my last four posts and the word Chimeric in the world of molecular biology refers to the fact that the gene is actually a combination of other genes or gene segments brought together from different sources. You may note that the concerns from previous attempts at using retroviruses are mitigated here because the transfection actually occurs, effectively, in a petri dish rather than in the body of the patient. Further, using a cytotoxic T cell is preferable to say antibody treatment since rather than relying on an orchestrated immune response to the antibody binding of its ligand to kill a target cell; cytotoxic T cells kill directly and serially.

There are essentially three steps in the engineering of a gene which encodes the CAR. The first is to identify a molecule, the new receptor’s ligand, that exists on the surface of the cells that are to be destroyed. The second is to find a molecule that has binding specificity for that ligand, perhaps a variable region from an antibody or the extracellular region of a co-stimulatory receptor.

For example Herceptin is a monoclonal antibody used in the treatment of breast and other cancers. It has binding specificity for erbB2 which is highly expressed on the surface of malignant cells. Another example comes from the fact that the CD8 receptor found on T Cells happens to bind with high affinity to the glycoprotein GP120 which is present on the envelope of HIV.

If an antibody is chosen, segments of the genes that encode the variable regions of the heavy and light chains (see previous posts) are linked together from the 3’ end of the light chain to the 5’ end of the heavy (note that DNA molecules are formed in one direction 5’ to 3’)₃. This produces a gene which encodes a single protein designated scFab or Single Chain Antigen Binding Fragment.

The next step is to find a suitable signal generation protein chain for the intracellular domain of the chimeric receptor. This step is far simpler than the first two given that it appears that there a relatively small number which can initiate T cell effector programs such as cytotoxicity. Second generation CARs, for example, used the zeta ( ζ) chain of the T Cell Receptor Complex joined to the scFab via the transmembrane region of a co-stimulatory molecule such as CD 8 or CD28₂. Third generation CARs use the intracellular signaling domains from 4-1 bb or OX40₂. I haven’t read of the reasoning for the change, but my thoughts are that it stems from the fact that 4=1 bb and OX40 are present only on T cells which are already activated. The ζ chain, on the other hand is involved in the initial activation of a T cell. Therefore the results of the signaling through 4-1 bb and OX40 would be more specific to cytotoxic effector function rather than the proliferative and cytokine release functions that are part of the in the initial activation of a T cell. This means that there wouldn’t be cascading bursts of T cell proliferation and cytokine production following each encounter with the CAR and it’s antigen thus lessening the potential for a massive expansion of the T cell population after injection and adverse reactions to very large releases of cytokines.

To illustrate the need for a signaling region, consider the ζ chain. The T and B Cell Antigen Receptors, whose formation I presented in my previous four posts, are key to the function of our adaptive immune response. However, they are not present in the membrane alone and they are not actually capable of producing an intracellular signal. Though I didn’t mention the other proteins in my posts, the Antigen Receptors are actually part of a complex which includes other proteins in close association which are capable because they contain ITAMs. The ITAMs; Immunoreceptor Tyrosine-based Activation Moieties, are initiators of stimulatory signals and are activated when the Antigen Receptor binds its ligand. In a T cell these proteins are collectively known as CD3 and together with the Antigen Receptor; the T Cell Receptor Complex.

Figure 1 – T cell Receptor Complex

Returning to our hero and the generation of the Chimeric Antigen Receptors that killed his cancer… In the lab (in vitro), the proliferation of his T Cells was induced, amazingly, using beads engineered to mimic the activity of dendritic cells which are the most proficient presenters of antigen to T cells in the body (in vivo). Then the genes encoding the third generation CAR are delivered to those cells, via a retrovirus. His T cells are subjected to a battery of quality tests and then cryogenically frozen until he was ready to receive them.

The scFab region of this CAR has binding affinity for a molecule (the receptor’s “ligand”) expressed on the surface of the malignant cells known as CD19. When the millions of his T Cells, rendered cytotoxic and specialized through synthetic bioengineering, were injected into his blood, the cancer was gone within 30 days.

Despite this astounding result, and the result of others in the trial, the work, is still far from complete. CD19 may be expressed on his malignant cells, but those cells were B cells, and CD19 is expressed on virtually all B Cells. So at 6 months, while his bone marrow was clear of the malignant cells, it was also clear of any B cells. While this was actually a big step to the positive given immunosuppressive chemo isn’t usually so selective, an “on-target but off-organ” effect has been seen in this and other studies₂. This occurs when the target of the anitgen targeted by the T cells is expressed on healthy cells not intended as targets. In the case of CD19, this is less an issue because the Immune System is regenerative and its function is specific. However, if the target is expressed on the cells of healthy tissue to a high enough degree, the results can be devistating.

The people included in the trail, as I interpret the information, had a very poor prognosis with advanced, chemorefractory cancers. The two fatalities in another CAR trial involved several differentiating factors, but the exact cause of death was presented as inderterminate. In one case there was the possibility of an unrelated infection as the cause, but in the other “on-target, off-organ” seemed likely. I will present more information on this as well as the “on-target but off-organ” effect in my next post.

* Note: I’m not a researcher nor a clinician, and nothing I present in this post, or any other post I may make on a health science topic, should in any way be considered medical advice, the intent of the authors of any of the texts or research I reference, or an authoritative presentation on any source used. This post is solely my understanding along with, perhaps, my ideas based on my understanding of the material I’ve read.

1. “Adoptive T Cell Therapy: Entering the era of Synthetic Biology”
Carl June
University of Pennsylvania
http://videocast.nih.gov/launch.asp?16471

2. “Safer CARs”
Helen E Heslop
Molecular Therapy (2010) 18 4, 661–662. doi:10.1038/mt.2010.42

3. “Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the y or C subunits of the immunoglobulin and T-cell receptors”
ZELIG ESHHAR*, TOVA WAKS, GIDEON GROSSt, AND DANIEL G. SCHINDLER
Proc. Natl. Acad. Sci. USA
Vol. 90, pp. 720-724, January 1993
Immunology

4. “HER 2/neu protein expression in colorectal cancer”
http://www.biomedcentral.com/1471-2407/6/123
B Schuell (a) , T Gruenberger (b) , W Scheithauer (a) , Ch Zielinski (a) and F Wrba (c)
a. Department of Internal Medicine I, Division of Clinical Oncology, University Hospital, Vienna, Austria
b. Department of Surgery, University Hospital, Vienna, Austria
c. Department of Pathology, University Hospital, Vienna, Austria

5. “Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the γ or ζ subunits of the immunoglobulin and T-cell receptors”
ZELIG ESHHAR*, TOVA WAKS, GIDEON GROSSt, AND DANIEL G. SCHINDLER
Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel
Proc. Natl. Acad. Sci. USA
Vol. 90, pp. 720-724, January 1993
Immunology

So you’ve been told that all the cells in your body contain the same genetic material? I’ve seen it written as a general principle of Genetics and a quick search around the web will yield many sources indicating that all somatic cells contain the exact same DNA. I did find a few articles in science magazines which “revealed” that this may not be the case, but the general consensus that they do was prevalent.

From all I’ve read, research and texts included, the DNA contained in all somatic cells is the same with one exception; the hematopoietic cells. These cells are the cells of the blood and they all descend from a single progenitor, the Human Hematopoietic Stem cells which can be found in bone marrow. These stem cells give rise to a diverse population of progeny including erythrocytes (red blood cells), T Cells, B Cells, Macrophages, and Natural Killer cells. To be accurate, the stem cells and their immediate descendants contain “germ-line” DNA, which is DNA that was formed during the individual’s fertilization event and is shared with virtually every other somatic cell in the body. However, as the hematopoietic descendants start to specify into the progenitors of T and B cells or “lymphopoietic” progenitors, the process of Somatic Recombination which creates the antigen receptors I’ve been writing about cuts out and discards parts of certain chromosomes thus creating genetically distinct human cells within our own bodies.

In my last post I presented the gene loci on which this process acts and highlighted the first actors in the process; the Recombination Activating Gene 1 & 2 proteins (RAG1 & RAG2). The arrangement of the gene loci is very important in this discussion and the rest of what I’m about to present doesn’t make much sense without this knowledge, so you can click this link to read that post if necessary. The process is performed by the V(D)J Recombinase and the process progresses as the molecules that make it up are assembled, I will introduce them as I describe the process.

It begins with two molecules each containing one RAG1 and one RAG2. These complexes then randomly bind, one each, to two of the hundreds of Recombination Signaling Sequences (RSSs) found surrounding each gene segment in the target antigen locus. It should be noted that the selection is not random across the segment. For example, two V regions will not join; it must be a V and J, for example, in a light chain locus. After binding, the two complexes come together and bind resulting in the formation of a loop of DNA between them which contains all the gene segments that may have been between the two segments brought together.

With the loop formed, the RAG complex will cut the strand exactly at the points where the RSS ends and the coding segment begins (see diagram below). This happens because the RAG complex has an inherent ability to act as an endonuclease. This essentially means that it is a protein that can break a DNA molecule into parts. This is the action that excises a piece of our DNA which is called the signal joint (because the RSS of each segment are joined flush). At this point, because of reasons relating to the chemical properties of the structure of a molecule of DNA, the ends of each strand which are cut fuse to form hairpins.

At this point other proteins join the complex to complete the recombinase and the process. These proteins are actually ubiquitous in human cells and I believe they are present for every mitotic and meiotic event that occurs over an individual’s lifetime. The first two proteins to bind to the growing V(D)J Recombinase are named Ku70:80 and they hold together both the ends of the chromosomal DNA strand, and those of the signal joint. Note, that this is the last I will mention the signal joint as once it’s ends are fused, it is no longer part of our story.

It is here where the process becomes a little unnerving for me. The very precise cut made by the RAG1/RAG2 complex to setup the joining of gene segments is one thing, but what happens next, to me, is another. The following video presents an excellent visualization of the entire process with a voice-over by Dr. Julie Theriot; a brilliant mind. I will, however, go into a little more detail below.

A protein named DNA-PK, or DNA-dependent Protein Kinase, is formed when another protein named DNA-PKcs binds tightly with Ku after the strands are bound. And to this another protein named Artemis joins. Artemis is also an endonuclease without which we would be ending our story with a broken strand of DNA. Artemis cuts the hair pins formed after RAG1/RAG2 excised the signal joint, but the location of its cut appears to be random. It has been found that they occur anywhere along the hair pin and this is where the fun starts.

With the location being random, unequal ends are often left which must be equalized before the DNA can be rejoined. Enter another protein that is found in all our cells and has a great name; Terminal Deoxynucleotidyl Transferase (TdT). TdT randomly adds bases to the cut ends resulting in what is essentially a newly formed random sequence on each. At the same time, two more newcomers bind to each other and then the Recombinase; DNA Ligase IV and XCR44. There are ubiquitous DNA repair proteins which and as soon as they bind, they start trying to re-join the strands; the name Ligase actually comes from the Latin “ligare” meaning “to bind or tie”. With TdT inserting random bases, the repair/ligation complex may need to knock out and replace non-complementary pairings such as a Guanine to a Thiamine.

With the ligation effort winning out in the end and the chromatid now back in one piece, the process of Somatic Recombination is complete. With just a few more steps we have a complete gene which, when transcribed will form one of the two chains in an Immunoglobulin. The process, however, has left the chromatid irrevocably changed; it is unique, different from any other member of its chromosome pair in the body, including other B cells. I’d like to note again that this change is key to the efficacy of our adaptive immune response. Without the enormous diversity generated by this process, neither the B and T cell antigen receptors nor the antibodies secreted by the former would be capable of binding effectively to the diverse and ever changing peptides making up evolving pathogens. However, it also introduces a potential for a number of pathologies, including cancer.

It has been shown in research1 that if the DNA repair mechanisms like DNA Ligase IV\XCR44 and the DNA damage checkpoints are not functioning properly, Somatic Recombination can result in several pathologies. These include aneuploidy, an abnormal number of chromosomes, translocations, where one part of a chromosome is fused with another, and neoplastic transformation, cancer.

In fact, according to both Janeway’s Immuno Biology (p 312) and a paper published in Nature Immunology in 20071, translocations are found in most lymphoma tissues. What is compelling for me about these translocations is that they most often involve what are known as proto-oncogenes which are genes encoding proteins that, when not functioning properly, cause cancer.

What causes DNA checkpoints and the DNA repair proteins to malfunction? I’ve seen the research showing the obvious conclusion; mutations forming in the genes coding for the proteins which carry out the functions. However, I suspect epigenetics may play a role in at least some of the scenarios. What causes these translocations? Given the fact that many lymphomas are age onset, I would conclude that probability must be considered, but the translocations of proto-oncogenes suggest to me that epigenetic factors should be examined. There are genes such as Myc (Myelocytomatosis)that regulate the expression of up to 15% of our entire genome and do so through epigenetic factors such as HAT (Histone Acetyltransferase). It should not be surprising that Myc is an oncogene and it is, in fact, one that is involved in the translocations found in lymphoma tissues.

1. Nature Immunology 8, 801 – 808 (2007)
Published online: 19 July 2007 | doi:10.1038/