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

It’s been my experience that when bacteria are discussed, it is usually in a negative light. This is despite the fact that there are ten times more bacterial cells in and on our bodies than human cells, and the co-location is actually mutually beneficial. Understandably, viruses get the same treatment, but with their potential sequestering for genetic therapies perhaps there will be a change. I have actually been thinking that viruses may have played a key role in early the evolution of life, but that is another story. This story, the one on somatic recombination, is a very complicated one, so you can review the first two parts, here, and here.  Note that I’m focusing on B cells, but the process is essentially the same in T Cells.

Given negative light in which viruses are seen, it seems ironic then that the mechanism by which a retrovirus integrates its genetic material into the infected cell is very similar to the mechanism by which our B and T cells produce the molecules needed to recognize and defeat bacterial and viral infections ₁. Two proteins, named RAG1 and RAG2, which start off the process in our cells, have been shown to form what is known as a transposase. In retroviruses such as the one which causes AIDS, a transposase is used to splice the viral genetic material into that of the infected cell. RAG1 & 2 combined in a human cell can do the exact same thing₂.

Even the genes which encode these proteins are extraordinary. Mammalian genes usually contain regions called introns which are non-coding and though transcribed, are edited out before the RNA is translated into a protein. However, the RAG genes, like most bacterial genes, contain none. This seems to me, at least, to be a very interesting evolutionary story. How did genes that don’t contain introns and code for proteins which form a transposase end up in our genome?  I’ll have to note this question for another post.

As I mentioned in my last post, the immunoglobulins that Recombination creates have a both a variable (V) regions and a constant (C) regions. They are made up of two identical pairs of protein strands, called chains, with each pair consisting of one light and one heavy (the heavy chains are the longer chains in the image to the right). It is the variable regions we are concerned with here and RAG 1 & 2 proteins join, and then incorporate a few other proteins to form what is known as VDJ recombinase to produce them. The recombinase is the lead actor in somatic recombination.

Before we continue to the process, the genes which code for the light and heavy chains are uncommon as well and therefore worth visiting. Ordinarily, the nucleotide sequence of a given gene is transcribed from the chromosome onto an RNA molecule that is then translated by a ribosome to produce the protein product. The location on a chromosome at which any given gene is located is known as the locus (loci, Pl.)

This is not the case, however, at the loci which encode the heavy and light chains of an Immunoglobulin. An examination of the light chain locus on chromosome 2 will reveal not a single gene but rather a large number of gene segments. Further, there are special sequences of nucleotides to be found surrounding those segments. First, up are the so-called Leader sequences which come into play after translation and mark an immunoglobulin for transport to the plasma membrane. Second are the even more interesting Recombination Signaling Sequences (RSSs) which are the markers to which the RAG 1 and RAG 2 complex binds.

As depicted in the diagram below, there are actually different types of gene segments within the locus which will be combined together in the process. The variable region generated by the VDJ recombinase will include one each of the V, D (heavy chain loci only), and J segments. The C segments code the constant regions and we’ve only found a handful so far. Estimates that I’ve read or heard for the number of V segments, on the other hand, range anywhere from forty to hundreds within a given locus.

The process of somatic recombination forming a Light Chain selects at random a V segment and a J segment and joins them together along with a C segment to form a single gene. The “D” in VDJ refers to the D, or Diversity, segments which exist only in Heavy Chain loci, in fact the proper spelling is V(D)J recombinase.  It is the process of joining the segments that produces the infinite potential for sequence recognition, not the segments themselves.  This process, Somatic Recombination,  is simple in concept, yet complex in execution and within, there is yet another twist which demonstrates the beauty as well as the danger in this amazing biological mechanism. In my next post, the process…

1.Janeway’s Immuno Biology
Seventh Edition
Garland Science

2. Nature. 1998 Aug 20;394(6695):744-51.
Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system.
Agrawal A, Eastman QM, Schatz DG.
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.