Application of dual affinity retargeting molecules to achieve optimal redirected T-cell killing of B-cell lymphoma

Abstract

IntroductionTargeted mAb-based therapies provide effective and safe treatments for hematologic malignancies. Rituximab, which specifically targets the B-cell antigen CD20, has had the greatest success, revolutionizing the treatment of the 2 most common forms of nonHodgkin lymphoma: follicular and diffuse large B-cell lymphoma. In addition, mAb-based therapies targeting CD52 (alemtuzumab) and CD33 (gemtuzumab ozogamicin) have been approved for the treatment of chronic lymphocytic leukemia and acute myelogenous leukemia, respectively. Despite the progress of these strategies, they do have limitations. Only a fraction of patients respond to rituximab, and the majority of those who do respond will eventually relapse. Treatment with alemtuzumab and gemtuzumab are limited by safety concerns, and many additional hematologic malignancies do not respond to treatment with any of these targeted therapies. Various therapies based on alternate mAbs, including second-generation anti-CD20 mAbs and those targeting alternate cell-surface proteins such as CD19, CD22, CD30, CD37, CD40, and CD74, have been developed and are at different stages of clinical testing in the hopes of providing approaches to treating a broader spectrum of hematologic malignancies that are poorly served by existing therapies. 1,2 Whereas targeting of cell-surface antigens themselves can mediate antitumor activity through the induction of apoptosis, most mAb-based activity against hematologic malignancies is reliant on either Fc-mediated effector functions such as complementdependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity 3,4 or is engineered through the conjugation of an immunotoxin or radiolabeled isotope. 1 Considering the potential of naturally occurring CTLs to mediate cell lysis, various strategies have also been explored to recruit CTLs to mediate tumor cell killing. Tumor-specific CTLs exert extremely potent effects through recognition of the corresponding peptide/MHC complex recognized by their TCR, and are among the most potent cells that mediate antitumor effects. A major limitation in generating tumorspecific CTLs in vivo is that their induction requires the use of vaccine strategies, such as dendritic cell-based vaccines, 5 that are capable of breaking tolerance to cancer self-antigens. One alternative is ex vivo expansion and activation of rare, tumor-specific CTLs for reinfusion into cancer patients. 6 However, cancer cells can down-regulate MHC expression as an escape mechanism, thus preventing the ability of CTLs to recognize their antigenic peptide. The genetic manipulation of patients' T cells to express chimeric antigen receptors comprising a tumor-specific antigen and T cellactivating properties before their adoptive transfer provides a non-MHC-restricted approach to targeting cancer, as was shown recently in the treatment of lymphoma with T cells engineered to recognize CD19. 7 However, the patient-specific manipulation and risk associated with this procedure represent major limitations to its expanded use. Alt...

A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19+tumor cells

“…The significant increase in avidity of the TandAb indicates that each domain is competent to bind its cognate target. K D of the CD19 HD37 /CD3 TR66 BiTE to CD19 and CD3 were 2.1 nM and 110 nM, respectively, and consistent with previous data, 21,22 indicating a substantially lower affinity for CD3 than AFM11-His. Finally, the SPR experiments suggest the possibility of simultaneous binding to the CD19 and CD3 antigens, which is a requirement for the mode-of-action of the TandAb.…”

“…In an assay of the contributions of distinct T cell populations to cytotoxicity, at the early time point (4 h) CD8 C T cells are the major contributors, whereas at the later time point (23 h) the contributions from CD8 C and CD4 C T cells are approximately equal; this is consistent with previous observations. 21,22 Finally, we observe that AFM11 potency is not correlated with target cell CD19 density.…”

“…Similarly, CAR T cells are expected to persist in patients for extended periods of time, and thus could also present significant risk in case of treatment-related complications. We rationalized that a T cell-recruiter that both possesses a longer t 1/2 than the BiTE, but not as long as a full-length IgG, and maintains a rigid structure that may be more favorable for optimal target engagement, 21 and thus increased cytotoxicity, would be optimal for dosing and the management of adverse events. Furthermore, though response to blinatumomab is impressive, with 43% of relapsed and refractory ALL patients achieving complete response, 9 a higher response rate is needed.…”

“…SPR measurements for binding to recombinant huCD19-Fc and huCD3ge; CD19 HD37 /CD3 TR66 BiTE was measured employing the same protocol as described. 21 The tabulated dissociation constant (K D ), association rate constant (k a ), and dissociation rate constant (k d ) were calculated using the global fit and the 1:1 Binding model across 6 concentration curves (0, 12.5, 25, 50, 100 and 200 nM) for AFM11-His and both comparator molecules tumor necrosis factor (TNF), and interferon (IFN)-g ( Fig. 6A) in the presence of CD19 C cells, but not in B cell-depleted PBMC or enriched T cell cultures.…”

“…To facilitate the comparison of the properties of the CD19 HD37 /CD3 TR66 BiTE with those presented in the literature, the SPR protocol described 21 …”