There are multiple clinical trials, assessing the efficacy of autologous bone marrow mononuclear cells in acute myocardial infarction. With a recent results of the TIME study, all 3 clinical trials (FOCUS, LateTIME and TIME), supported by US Federal Government and coordinated by Cardiovascular Cell Therapy Research Network (CCTRN), considered as failed. Everyone today is discussing the possible reasons of failures. It could be trial design, end points, statistical analysis, cell source and cell product preparation. Maybe cells simply don’t work! In this post I’ll try to dig in possible issues with cell product characterization and preparation for CCTRN trials. All things, that I’m going to discuss here, have been published in literature.
Autologous bone marrow mononuclear cells (MNC) were used in all CCTRN trials. Some professionals think that autologous bone marrow is a wrong starting material for manufacturing an efficacious product for heart attack patients. Allogeneic bone marrow cells from young healthy volunteers could be more advantageous in terms of functionality. The hint to possible advantage of allogeneic cells was provided by recent POSEIDON trial, published in parallel with the TIME study. Allogeneic or autologous myocardium is another promising cell source. Cardiac progenitor cells demonstrated good safety profile and now moving to Phase 2 of trials (see CADUCEUS and SCIPIO).
Cell population selection and stem cell enrichment
Some folks argue that “crude” cell product (bone marrow MNC) will provide less (or no) therapeutic benefit compare to more sophisticated – selected and purified cell populations. CCTRN cell products are “crude”. Cell processing doesn’t involve selection of specific populations and cell culture. Some examples of more sophisticated cell products, which undergo clinical trials, may include: purified CD34+ or CD133+ cells, ex vivo expanded mesenchymal stromal cells, c-Kit+ cardiac progenitor cells.
The purity of progenitor cell populations in TIME trial were: 2.2% of CD34+ and 1.1% CD34+/CD133+.
MNC isolation procedure
MNC could be isolated by density gradient centrifugation. Ficoll-based density media is commonly used for this purpose. The technique was not automated until now and involved open system processing. In CCTRN trials MNC were isolated by automatic method, using cell processing device Sepax (BioSafe). Automation of MNC isolation allows GMP-compliant closed system processing and minimization of difference between facilities.
In the commentary to TIME trial results, Eduardo Marbán wrote:
The lack of benefit of MNC therapy in TIME and in the other CCTRN studies raises 2 possibilities: MNCs do not work, in which case the previous positive clinical studies were red herrings, or the CCTRN trials used an inactive cell product. The latter possibility cannot be discounted because MNCs isolated by the automated system used in this study have not been tested in an animal model of MI, and cell bioactivity has not been verified in any human studies of heart disease.
If we can take this argument and agree that manual method allows better cell function, it still can’t solve a problem of heterogeneity of cell preparations and, as a consequence, mixed trials results (see REPAIR-AMI versus ASTAMI).
Sepax was not validated in animal model of heart attack, but the authors mentioned (without a link) some study on hind limb ischemia:
In vitro and in vivo studies comparing the delivery of Sepax-derived BMCs with that of open Ficoll-selected BMCs demonstrated phenotypic equivalence and equal efficacy on hind limb recovery in a murine model of hind limb perfusion.
Results of clinical validation of Sepax were published by CCTRN here.Validation revealed significant total nucleated cell loss (median recovery 23.7%). Previously, Sepax was compared with manual ficoll gradient separation. The recovery of MNC after Sepax was greater than after manual ficoll.
Contamination by red blood cells
Significant number of red blood cells (RBC) in cell product can negatively affect the function of bone marrow MNC, derived from heart attack patients. Seem like it was not the issue in CCTRN trials, because the authors didn’t notice any significant contamination by RBC (see quote below).
Heparin could be used as anticoagulant during the processing and in the final product. It was shown that heparin can affect function of bone marrow MNC and, possibly, influence a cell recovery. CCTRN cell preparation involved some amount of heparin in final product, but the authors noted:
The cell product was devoid of significant red blood cell contamination, contained only minuscule amounts of heparin (0.1 U/mL), and most participants were infused within 1 hour of completion of cell processing,(11) thereby avoiding concerns recently expressed in the literature.
Total nucleated cell number and CD34+ cell dose
One of the issues could be low total MNC or progenitor cell number injected. About 150 millions of total MNC were injected in TIME trial settings. 2.2% corresponds to 3.3 million of CD34+ cells. Is it enough or too low? Based on meta-analysis of all published trials, there is no clear correlation between total MNC number and efficacy. However, some studies highlight the significance of CD34+ cell dose. For example, Amorcyte Phase 1 trial, concluded:
No patient experienced a deterioration in heart muscle function who received 10 million cells or more whereas 30 to 40 percent of patients not receiving a therapeutic dose did.
So, basically, some folks are trying to define a threshold of CD34+ cell dose at 10 millions to see any benefit. The number of CD34+ in TIME trial cell preparations was less than 5 millions. Well, If recovery of MNC by Sepax was so low (23.7%), the TIME trial didn’t allow to get close to 10 million of CD34+.
Cell product composition
I think, it’s important to know the final cell product composition. Not just % of stem/ progenitor cell populations and RBC, but the whole composition – CD3, CD14, CD90, CD33… Based on Sepax validation data, median % of lymphocytes, monocytes and granulocytes were 42.5, 8.0 and 39.0 respectively. We don’t know how composition of immune accessory cells will influence the outcome, but it will be interesting to compare this variable across the trials.
Cell culture, storage, logistics
I’m excluding these issues from analysis, because the cells in CCTRN trials were processed and administered at point of care withing few hours. However, these issues must be considered in analysis of cell preparations in other trials.
Now, I’d like to ask you:
(1) Do you think problematic cell prep was a reason for CCTRN trials failure?
(2) If yes, what component of cell prep was failed and what can we learn for it?
(3) Do you think cell prep was fine and the reason of failure is simple – cell do not work?