Treatment of acute myeloid leukemia, an aggressive hematopoietic malignancy of myeloid progenitors, has remained rather stagnant over the course of several decades. Infusions of cytarabine and anthracycline antibiotics have dominated the landscape of AML therapy, with minor changes to dosing schedule occasionally making slight adjustments to efficacy or tolerability. Improvements in prognosis have been bittersweet, with most progress seen in younger populations less likely to get the disease, and already more likely to achieve remission and to meet survival milestones. Much of this progress is attributed to other factors, such as improved supportive care and availability of hematopoietic stem cell and platelet transfusion. In most patients, occupying the 60-and-above demographic, improvements in survival have not been significant. In turn, the population impact of AML has changed little over time. While accounting for about one-third of total leukemia cases and one percent of total cancer cases, AML accounts for about one half of total leukemia deaths and two percent of total cancer deaths.
Most advances straying away from standard treatment have been in important pathways that could be impactful in subsets of the overall AML patient population. Tyrosine kinases are implicated in numerous cancers including AML, with activity-enhancing mutations conferring growth advantages to malignant cells. About one-third of AML patients have mutations in one such kinase, FLT3, and may benefit from inhibitors to tyrosine kinases overall and from FLT3- specific agents. Mutations in isocitrate dehydrogenases highlight another subpopulation, about one-fifth of AML patients, who might benefit from emerging agents that inhibit these pathways from creating a leukemia-favoring environment in the bone marrow. Other pathways similarly implicated in numerous cancers including AML are being targeted with new agents that can benefit some AML patients, such as Hedgehog signaling and apoptotic regulation. Still, breakthroughs are needed that can help most AML patients, particularly in the cases of relapsed leukemia that occurs in most patients within a year or two after remission is achieved. CD33 is among a few molecular targets for AML, though it is just as ubiquitously expressed on healthy myeloid cells. Antibody-drug conjugates like Mylotarg have made progress in this approach, though hematopoietic toxicities have made treatment difficult in older populations. Clever techniques such as ablation of CD33 from healthy myeloid progenitors may be supportive in CD33-based approaches, and immunotherapy involving CD33-targeting is a rapidly growing research focus.
This dissertation describes a new type of bispecific antibody that binds CD33 on AML and CD3 on cytotoxic T cells in a proof-of-concept study. Various formats for bifunctional molecules have been created and used clinically, including antibody-drug conjugates and bispecific antibodies that simultaneously engage antigens on two different types of cells. Those like the one described here, bispecific T-cell engagers, have typically taken the form of single-chain fusion proteins containing the variable regions binding to both antigens of interest. Other bispecific antibodies have imitated naturally-occurring immunoglobulin structures, boasting superior pharmacokinetics while facing steep obstacles in large-scale production. The single-chain fusions, easier to produce, can face difficulties in full engagement, with loss of function sometimes seen in fusion partners at the C-terminus.
We propose a new format, believed to present two antigen-binding domains in N-terminal positions on a two-chain heterodimeric structure. Capitalizing on an elegantly designed system of hydrophobic cores and hydrogen-bonding networks generating an orthogonal heterodimer, we added an immunoglobulin hinge region to secure a permanently-bound heterodimer, and attached domains binding to CD3 and CD33. We hypothesized that this design, ensured to present its antibody components at N-termini, could bind two antigens at a distance appropriate for facilitating T cell cytotoxicity to AML.
After expressing and purifying these proteins in mammalian cells, we demonstrated their ability to persist as a bispecific heterodimer. We showed in vitro that our bispecific heterodimers could bind both CD3+ and CD33+ cells, and that they bolstered T cell cytotoxicity to AML cell lines in a dose-dependent manner. Monomeric components bound only CD3+ or CD33+ cells depending on antibody variable domain present, and had no effect on T cell cytotoxicity. In a mouse model of minimal residual disease, T cells alone did not have a significant effect on the growth of AML, nor did they have an effect on overall survival. T cells with bispecific heterodimer greatly extended survival, and mice of this treatment group were free of leukemia.
These findings suggest that this format for bispecific proteins allows for robust simultaneous engagement with both antigens of interest in a manner conducive to T cell cytotoxicity against AML. We believe this presents a compelling modular system for bispecific antibodies, where CD3- and CD33-binding domains can be readily swapped with domains binding to other cancer- or immune cell-specific antigens, and can be further developed into a trispecific system engaging other immune cells or extending half-life with anti-albumin or Fc domains.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/x2n3-0f63 |
| Date | January 2022 |
| Creators | Burke, Alan Austin |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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